Bispecific binding molecules binding to vegf and ang2

ABSTRACT

Bispecific binding molecules binding to both VEGF and Ang2, preferably in the form of immunoglobulin single variable domains like VHHs and domain antibodies, pharmaceutical compositions containing the same and their use in the treatment of diseases that are associated with VEGF- and/or Ang2-mediated effects on angiogenesis are disclosed. Further, nucleic acids encoding bispecific binding molecules, host cells and methods for preparing same are also described.

FIELD OF THE INVENTION

The invention relates to the field of human therapy, in particularcancer therapy and agents and compositions useful in such therapy.

BACKGROUND OF THE INVENTION

When tumors reach a critical size of approximately 1 mm³ they becomedependent on angiogenesis for maintaining blood supply with oxygen andnutrients to allow for further growth. Anti-angiogenesis therapies havebecome an important treatment option for several types of tumors. Thesetherapies have focused on blocking the VEGF pathway (Ferrara et al., NatRev Drug Discov. 2004 May; 3(5):391-400.) by neutralizing VEGF (Avastin)or its receptors (Sutent and Sorafinib). Recent studies in mice haveshown, that Angiopoietin2 (Ang2), a ligand of the Tie2 receptor,controls vascular remodeling by enabling the functions of otherangiogenic factors, such as VEGF. Ang2 is primarily expressed byendothelial cells, strongly induced by hypoxia and other angiogenicfactors and has been demonstrated to regulate tumor vessel plasticity,allowing vessels to respond to VEGF and FGF2 (Augustin et al., Nat RevMol Cell Biol. 2009 March; 10(3):165-77.). Consistent with this role,the deletion or inhibition of Ang2 results in reduced angiogenesis (Galeet al., Dev Cell. 2002 September; 3(3):302-4.) (Falcón et al., Am JPathol. 2009 November; 175(5):2159-70.). Elevated Ang2 serumconcentrations have been reported for patients with colorectal cancer,NSCLC and melanoma (Goede et al., Br J Cancer. 2010 Oct. 26;103(9):1407-14), (Park et al., Chest. 2007 July; 132(1): 200-6.),(Helfrich et al., Clin Cancer Res. 2009 Feb. 15; 15(4):1384-92.). In CRCcancer Ang2 serum levels correlate with therapeutic response toanti-VEGF therapy.

The Ang-Tie system consists of 2 receptors (Tie1 and Tie2) and 3 ligands(Ang1, Ang2 and Ang4) (Augustin et al., Nat Rev Mol Cell Biol. 2009March; 10(3):165-77.). Tie2, Ang1 and Ang2 are the best studied membersof this family, Tie1 is an orphan receptor and the role of Ang4 forvascular remodelling still needs to be defined. Ang2 and Ang1 mediateopposing functions upon Tie2 binding and activation. Ang2-mediated Tie2activation results in endothelial cell activation, pericytedissociation, vessel leakage and induction of vessel sprouting. Incontrast to Ang2, Ang1 signaling maintains vessel integrity byrecruitment of pericytes, thereby maintaining endothelial cellquiescence.

Angiopoietin 2 (Ang2) is a secreted, 66 kDa ligand for the Tie2 receptortyrosine kinase (Augustin et al., Nat Rev Mol Cell Biol. 2009 March;10(3):165-77.). Ang2 consists of an N-terminal coiled-coil domain and aC-terminal fibrinogen-like domain, the latter is required for Tie2interaction. Ang2 is primarily expressed by endothelial cells andstrongly induced by hypoxia and other angiogenic factors, includingVEGF. Tie2 is found on endothelial cells, haematopoietic stem cells andtumor cells. Ang2-Tie2 has been demonstrated to regulate tumor vesselplasticity, allowing vessels to respond to VEGF and FGF2.

In vitro Ang2 has been shown to act as a modest mitogen, chemoattractantand inducer of tube formation in human umbilical vein endothelial cells(HUVEC). Ang2 induces tyrosine phosphorylation of ectopically expressedTie2 in fibroblasts and promotes downstream signaling events, such asphosphorylation of ERK-MAPK, AKT and FAK in HUVEC. An antagonistic roleof Ang2 in Ang1-induced endothelial cell responses has been described.

Ang2-deficiency has been shown to result in a profound lymphaticpatterning defect in mice. Although the loss of Ang2 is dispensable forembryonic vascular development, Ang2-deficient mice have persistentvascular defects in the retina and kidney. Together with the dynamicpattern of Ang2 expression at sites of angiogenesis (for example ovary),these findings indicate that Ang2 controls vascular remodeling byenabling the functions of other angiogenic factors, such as VEGF.

The Ang2-Tie2 system exerts crucial roles during the angiogenic switchand later stages of tumor angiogenesis. Ang2 expression is stronglyup-regulated in the tumor-associated endothelium. Reduced growth oftumors has been observed when implanted into Ang2-deficient mice,especially during early stages of tumor growth. Therapeutic blocking ofAng2 with Ang2 mAbs has shown broad efficacy in a variety of tumorxenograft models. Additive effects of Ang2 mAbs with inhibitors ofVEGFR2 (mAbs and small molecular weight inhibitors) have been described.

As described in e.g. US2008/0014196 and WO2008/101985, angiogenesis isimplicated in the pathogenesis of a number of disorders, including solidtumors and metastasis as well as eye diseases. One of the most importantpro-angiogenic factors is vascular endothelial growth factor (VEGF),also termed VEGF-A or vascular permeability factor (VPF). VEGF belongsto a gene family that includes placenta growth factor (PIGF), VEGF-B,VEGF-C, VEGF-D, VEGF-E and VEGF-F. Alternative splicing of mRNA of asingle gene of human VEGF results in at least six isoforms (VEGF121,VEGF145, VEGF165, VEGF183, VEGF189, and VEGF206), VEGF165 being the mostabundant isoform.

Two VEGF tyrosine kinase receptors (VEGFR) have been identified thatinteract with VEGF, i.e. VEGFR-1 (also known as Fit-1) and VEGFR-2 (alsoknown as KDR or FIK-1). VEGFR-1 has the highest affinity for VEGF, whileVEGFR-2 has a somewhat lower affinity for VEGF. Ferrara (Endocrine Rev.2004, 25: 581-611) provide a detailed description of VEGF, theinteraction with its receptors and its function in normal andpathological processes can be found in Hoeben et al. Pharmacol. Rev.2004, 56: 549-580.

VEGF has been reported to be a pivotal regulator of both normal andabnormal angiogenesis (Ferrara and Davis-Smyth, Endocrine Rev. 1997, 18:4-25; Ferrara J. Mol. Med. 1999, 77: 527-543). Compared to other growthfactors that contribute to the processes of vascular formation, VEGF isunique in its high specificity for endothelial cells within the vascularsystem.

VEGF mRNA is overexpressed by the majority of human tumors. In the caseof tumor growth, angiogenesis appears to be crucial for the transitionfrom hyperplasia to neoplasia, and for providing nourishment for thegrowth and metastasis of the tumor (Folkman et al., 1989, Nature339-58), which allows the tumor cells to acquire a growth advantagecompared to the normal cells. Therefore, anti-angiogenesis therapieshave become an important treatment option for several types of tumors.These therapies have focused on blocking the VEGF pathway (Ferrara etal., Nat Rev Drug Discov. 2004 May; 3(5): 391-400.

VEGF is also involved in eye diseases. The concentration of VEGF in eyefluids is highly correlated with the presence of active proliferation ofblood vessels in patients with diabetic and other ischemia-relatedretinopathies. Furthermore, recent studies have demonstrated thelocalization of VEGF in choroidal neovascular membranes in patientsaffected by age-related macular degeneration (AMD). Up-regulation ofVEGF has also been observed in various inflammatory disorders. VEGF hasbeen implicated in the pathogenesis of rheumatoid arthritis, aninflammatory disease in which angiogenesis plays a significant role.

The elucidation of VEGF and its role in angiogenesis and differentprocesses has provided a potential new target of therapeuticintervention. The function of VEGF has been inhibited by small moleculesthat block or prevent activation of VEGF receptor tyrosine kinases(Schlaeppi and Wood, 1999, Cancer Metastasis Rev., 18: 473-481) andconsequently interfere with the VEGF receptor signal transductionpathway. Cytotoxic conjugates containing bacterial or plant toxins caninhibit the stimulating effect of VEGF on tumor angiogenesis. VEGF-DT385toxin conjugates (diphtheria toxin domains fused or chemicallyconjugated to VEGF165), for example, efficiently inhibit tumor growth invivo. Tumor growth inhibition could also be achieved by delivering aFlk-1 mutant or soluble VEGF receptors by a retrovirus.

VEGF-neutralizing antibodies, such as A4.6.I and MV833, have beendeveloped to block VEGF from binding to its receptors and have shownpreclinical antitumor activity (Kim et al. Nature 1993, 362: 841-844;Folkman Nat. Med. 1995, 1: 27-31; Presta et al. Cancer Res. 1997, 57:4593-4599; Kanai et al. Int. J. Cancer 1998, 77: 933-936; Ferrara andAlitalo Nat. Med. 1999, 5: 1359-1364; 320, 340. For a review oftherapeutic anti-VEGF approaches trials, see Campochiaro and Hackett(Oncogene 2003, 22: 6537-6548).

Most clinical experience has been obtained with A4.6.1, also calledbevacizumab (Avastin®; Genentech, San Francisco, Calif.).

WO2008/101985 describes immunoglobulin single variable domains fromcamelides (VHHs or “Nanobodies®, as defined herein) that bind to VEGF,and their use in the treatment of conditions and diseases characterizedby excessive and/or pathological angiogenesis or neovascularization.

It has been an object of the present invention to provide novelanti-angiogenic binding molecules for human therapy.

It has been a further object of the invention to provide methods for theprevention, treatment, alleviation and/or diagnosis of such diseases,disorders or conditions, involving the use and/or administration of suchbinding molecules and compositions comprising them. In particular, it ishas been an object of the invention to provide such pharmacologicallyactive binding molecules, compositions and/or methods that provideadvantages compared to the agents, compositions and/or methods currentlyused and/or known in the art. These advantages include improvedtherapeutic and/or pharmacological properties and/or other advantageousproperties, e.g. for manufacturing purposes, especially as compared toconventional antibodies as those described above, or fragments thereof.

BRIEF SUMMARY OF THE INVENTION

According to a first aspect, there are provided bispecific bindingmolecules, preferably bispecific immunoglobulins, preferablyimmunoglobulin single variable domains like VHHs and domain antibodies,which comprise at least one VEGF-binding component and at least oneAng2-binding component in a single molecule. Preferably, said bispecificbinding molecules further comprise a serum albumin binding component.

More specifically, a bispecific binding molecule of the inventionessentially comprises (i) a Ang2-binding component specifically bindingto at least one epitope of Ang2 and (ii) a VEGF-binding componentspecifically binding to at least an epitope of VEGF, wherein thecomponents are linked to each other in such a way that theysimultaneously bind to Ang2 and VEGF or that they bind to either Ang2 orVEGF at a time.

According to preferred aspects of the invention, the two componentscomprise one or more immunoglobulin single variable domains that may be,independently of each other, VHHs or domain antibodies, and/or any othersort of immunoglobulin single variable domains, such as VL domains, asdefined herein, provided that each of these immunoglobulin singlevariable domains will bind the antigen, i.e. Ang2 or VEGF, respectively.

According to a preferred embodiment, the immunoglobulin single variabledomains are of the same type, in particular, all immunoglobulin singlevariable domains are VHHs or domain antibodies.

According to a particularly preferred embodiment, all immunoglobulinsingle variable domains are VHHs, preferably humanized (or“sequence-optimized”, as defined herein) VHHs. Accordingly, theinvention relates to bispecific binding molecules comprising an(optionally humanized or sequence-optimized) anti-Ang2 VHH and an(optionally humanized or sequence-optimized) anti-VEGF VHH.

However, it will be clear to the skilled person that the teaching hereinmay be applied analogously to bispecific binding molecules includingother anti-Ang2 or anti-VEGF immunoglobulin single variable domains,such as domain antibodies.

In another aspect, the invention relates to nucleic acids encoding thebispecific binding molecules of the invention as well as host cellscontaining same.

The invention further relates to a product or composition containing orcomprising at least one bispecific binding molecule of the invention andoptionally one or more further components of such compositions.

The invention further relates to methods for preparing or generating thebispecific binding molecules, nucleic acids, host cells, products andcompositions described herein.

The invention further relates to applications and uses of the bispecificbinding molecules, nucleic acids, host cells, products and compositionsdescribed herein, as well as to methods for the prevention and/ortreatment for diseases and disorders that can be modulated by inhibitionof Ang2.

It has been found that the Ang2-binding component of the bispecificbinding molecules according to the present invention binds to andantagonizes Ang2 with a potency at least 5,000 times higher, preferably10,000 times higher than to Ang1 or Ang4. This will largely avoidblocking activation of Ang1-mediated signalling, which would counter theintended anti-angiogenetic effect.

It has further been found that the VEGF-binding component of thebi-specific binding molecules according to the present invention bindsto VEGF-A with an affinity of at least 1,000 times higher, preferebly atleast 5,000 times higher, more preferably at least 10,000 times higherthan to VEGF-B, VEGF-C, VEGF-D or PLGF. Due to the highly preferentialbinding to VEGF-A the signaling of VEGFR3, which modulates of lymphangiogenesis, is not interfered with.

In a preferred embodiment the bispecific binding molecules of thepresent invention are provided as linked VHH domains. Such molecules aresignificantly smaller than conventional antibodies and have thus thepotential for penetrating into a tumor deeper than such conventionalantibodies. This benefit is further accentuated by the specificsequences disclosed herein after being free of glycosylation sites.

Further, due to the bispecific nature (VEGF- and Ang2-binding componentsin one molecule) the tumor penetration of both functionalities will benecessarily equal, which will ensure that the beneficial effects of thecombined antagonism of VEGF and Ang2 will be provided within the wholedepth of penetration of the tumor. This is an advantage over thecombination of individual antagonists against these targets, since thedepth of penetration of individual antagonists will always vary to somedegree.

Another advantage of a preferred bispecific binding molecules of thepresent invention is their increased serum half-like due to a serumalbumin binding component such as a serum albumin binding molecule asdescribed herein.

These and other aspects, embodiments, advantages and applications of theinvention will become clear from the further description hereinbelow.

DEFINITIONS

Unless indicated or defined otherwise, all terms used have their usualmeaning in the art, which will be clear to the skilled person. Referenceis for example made to the standard handbooks, such as Sambrook et al,“Molecular Cloning: A Laboratory Manual” (2nd Ed.), Vols. 1-3, ColdSpring Harbor Laboratory Press (1989); Lewin, “Genes IV”, OxfordUniversity Press, New York, (1990), and Roitt et al., “Immunology”(2^(nd) Ed.), Gower Medical Publishing, London, New York (1989), as wellas to the general background art cited herein; Furthermore, unlessindicated otherwise, all methods, steps, techniques and manipulationsthat are not specifically described in detail can be performed and havebeen performed in a manner known per se, as will be clear to the skilledperson. Reference is for example again made to the standard handbooks,to the general background art referred to above and to the furtherreferences cited therein.

The term “bispecific binding molecule” refers to a molecule comprisingat least one Ang2-binding molecule (or “Ang2-binding component”) and atleast one VEGF-binding molecule (or “VEGF-binding component”). Abispecific binding molecule may contain more than one Ang2-bindingmolecule and/or more than one VEGF-binding molecule, i.e. in the casethat the bispecific binding molecule contains a biparatopic (as definedbelow) Ang2-binding molecule and/or a biparatopic VEGF-binding molecule,in the part of the molecule that binds to Ang2 or to VEGF, i.e. in its“Ang2-binding component” (or anti-Ang2 component) or “VEGF-bindingcomponent” (or anti-VEGF component), respectively. The word “bispecific”in this context is however not to be construed as to exclude furtherbinding components with binding specificity to molecules other than VEGFand Ang2 from the bispecific binding molecule. Non-limiting examples ofsuch further binding components are binding components binding to serumalbumin.

Unless indicated otherwise, the terms “immunoglobulin” and“immunoglobulin sequence”—whether used herein to refer to a heavy chainantibody or to a conventional 4-chain antibody—are used as general termsto include both the full-size antibody, the individual chains thereof,as well as all parts, domains or fragments thereof (including but notlimited to antigen-binding domains or fragments such as VHH domains orVH/VL domains, respectively). In addition, the term “sequence” as usedherein (for example in terms like “immunoglobulin sequence”, “antibodysequence”, “(single) variable domain sequence”, “VHH sequence” or“protein sequence”), should generally be understood to include both therelevant amino acid sequence as well as nucleic acid sequences ornucleotide sequences encoding the same, unless the context requires amore limited interpretation.

The term “domain” (of a polypeptide or protein) as used herein refers toa folded protein structure which has the ability to retain its tertiarystructure independently of the rest of the protein. Generally, domainsare responsible for discrete functional properties of proteins, and inmany cases may be added, removed or transferred to other proteinswithout loss of function of the remainder of the protein and/or of thedomain.

The term “immunoglobulin domain” as used herein refers to a globularregion of an antibody chain (such as e.g. a chain of a conventional4-chain antibody or of a heavy chain antibody), or to a polypeptide thatessentially consists of such a globular region. Immunoglobulin domainsare characterized in that they retain the immunoglobulin foldcharacteristic of antibody molecules, which consists of a 2-layersandwich of about 7 antiparallel beta-strands arranged in twobeta-sheets, optionally stabilized by a conserved disulphide bond. Animmunoglobulin domain comprises (a) variable domain(s), i.e., one ormore immunoglobulin variable domains.

The term “immunoglobulin variable domain” as used herein means animmunoglobulin domain essentially consisting of four “framework regions”which are referred to in the art and hereinbelow as “framework region 1”or “FR1”; as “framework region 2” or “FR2”; as “framework region 3” or“FR3”; and as “framework region 4” or “FR4”, respectively; whichframework regions are interrupted by three “complementarity determiningregions” or “CDRs”, which are referred to in the art and hereinbelow as“complementarity determining region 1” or “CDR1”; as “complementaritydetermining region 2” or “CDR2”; and as “complementarity determiningregion 3” or “CDR3”, respectively. Thus, the general structure orsequence of an immunoglobulin variable domain can be indicated asfollows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. It is the immunoglobulinvariable domain(s) that confer specificity to an antibody for theantigen by carrying the antigen-binding site. In the context of thepresent invention immunoglobulin single variable domains like VHHs anddomain antibodies are preferred.

The term “immunoglobulin single variable domain” as used herein means animmunoglobulin variable domain which is capable of specifically bindingto an epitope of the antigen without pairing with an additional variableimmunoglobulin domain. One example of immunoglobulin single variabledomains in the meaning of the present invention are “domain antibodies”,such as the immunoglobulin single variable domains VH and VL (VH domainsand VL domains). Another example of immunoglobulin single variabledomains are “VHH domains” (or simply “VHHs”) from camelids, as definedhereinafter.

In view of the above definition, the antigen-binding domain of aconventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgEmolecule; known in the art) or of a Fab fragment, a F(ab′)2 fragment, anFv fragment such as a disulphide linked Fv or a scFv fragment, or adiabody (all known in the art) derived from such conventional 4-chainantibody, would normally not be regarded as an immunoglobulin singlevariable domain, as, in these cases, binding to the respective epitopeof an antigen would normally not occur by one (single) immunoglobulindomain but by a pair of (associating) immunoglobulin domains such aslight and heavy chain variable domains, i.e. by a VH-VL pair ofimmunoglobulin domains, which jointly bind to an epitope of therespective antigen.

“VHH domains”, also known as VHHs, VHH domains, VHH antibody fragments,and VHH antibodies, have originally been described as the antigenbinding immunoglobulin (variable) domain of “heavy chain antibodies”(i.e. of “antibodies devoid of light chains”; Hamers-Casterman C,Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa E B, BendahmanN, Hamers R.: “Naturally occurring antibodies devoid of light chains”;Nature 363, 446-448 (1993)). The term “VHH domain” has been chosen inorder to distinguish these variable domains from the heavy chainvariable domains that are present in conventional 4-chain antibodies(which are referred to herein as “V_(H) domains” or “VH domains”) andfrom the light chain variable domains that are present in conventional4-chain antibodies (which are referred to herein as “V_(L) domains” or“VL domains”). VHH domains can specifically bind to an epitope withoutan additional antigen binding domain (as opposed to VH or VL domains ina conventional 4-chain antibody, in which case the epitope is recognizedby a VL domain together with a VH domain). VHH domains are small, robustand efficient antigen recognition units formed by a singleimmunoglobulin domain.

In the context of the present invention, the terms VHH domain, VHH, VHHdomain, VHH antibody fragment, VHH antibody, as well as “Nanobody®” and“Nanobody® domain” (“Nanobody” being a trademark of the company AblynxN.V.; Ghent; Belgium) are used interchangeably and are representativesof immunoglobulin single variable domains (having the structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and specifically binding to an epitopewithout requiring the presence of a second immunoglobulin variabledomain), and which are distinguished from VH domains by the so-called“hallmark residues”, as defined in e.g. WO2009/109635, FIG. 1.

The amino acid residues of a immunoglobulin single variable domain, e.g.a VHH, are numbered according to the general numbering for V_(H) domainsgiven by Kabat et al. (“Sequence of proteins of immunological interest”,US Public Health Services, NIH Bethesda, Md., Publication No. 91), asapplied to VHH domains from Camelids, as shown e.g. in FIG. 2 ofRiechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999).According to this numbering

-   -   FR1 comprises the amino acid residues at positions 1-30,    -   CDR1 comprises the amino acid residues at positions 31-35,    -   FR2 comprises the amino acids at positions 36-49,    -   CDR2 comprises the amino acid residues at positions 50-65,    -   FR3 comprises the amino acid residues at positions 66-94,    -   CDR3 comprises the amino acid residues at positions 95-102, and    -   FR4 comprises the amino acid residues at positions 103-113.

However, it should be noted that—as is well known in the art for V_(H)domains and for VHH domains—the total number of amino acid residues ineach of the CDRs may vary and may not correspond to the total number ofamino acid residues indicated by the Kabat numbering (that is, one ormore positions according to the Kabat numbering may not be occupied inthe actual sequence, or the actual sequence may contain more amino acidresidues than the number allowed for by the Kabat numbering). This meansthat, generally, the numbering according to Kabat may or may notcorrespond to the actual numbering of the amino acid residues in theactual sequence.

Alternative methods for numbering the amino acid residues of V_(H)domains, which methods can also be applied in an analogous manner to VHHdomains, are known in the art. However, in the present description,claims and figures, the numbering according to Kabat and applied to VHHdomains as described above will be followed, unless indicated otherwise.

The total number of amino acid residues in a VHH domain will usually bein the range of from 110 to 120, often between 112 and 115. It shouldhowever be noted that smaller and longer sequences may also be suitablefor the purposes described herein.

Immunoglobulin single variable domains, e.g. VHHs and domain antibodies,according to the preferred embodiments of the invention, have a numberof unique structural characteristics and functional properties whichmakes them highly advantageous for use in therapy as functionalantigen-binding molecules. In particular, and without being limitedthereto, VHH domains (which have been “designed” by nature tofunctionally bind to an antigen without pairing with a light chainvariable domain) can function as single, relatively small, functionalantigen-binding structural units.

Due to their unique properties, immunoglobulin single variable domains,as defined herein, like VHHs or VHs (or VLs)—either alone or as part ofa larger polypeptide, e.g. a biparatopic molecule—offer a number ofsignificant advantages:

-   -   only a single domain is required to bind an antigen with high        affinity and with high selectivity, so that there is no need to        have two separate domains present, nor to assure that these two        domains are present in the right spacial conformation and        configuration (i.e. through the use of especially designed        linkers, as with scFv's);    -   immunoglobulin single variable domains can be expressed from a        single nucleic acid molecule and do not require any        post-translational modification (like glycosylation;    -   immunoglobulin single variable domains can easily be engineered        into multivalent and multispecific formats (as further discussed        herein);    -   immunoglobulin single variable domains have high specificity and        affinity for their target, low inherent toxicity and can be        administered via alternative routes than infusion or injection;    -   immunoglobulin single variable domains are highly stable to        heat, pH, proteases and other denaturing agents or conditions        and, thus, may be prepared, stored or transported without the        use of refrigeration equipments;    -   immunoglobulin single variable domains are easy and relatively        inexpensive to prepare, both on small scale and on a        manufacturing scale. For example, immunoglobulin single variable        domains can be produced using microbial fermentation (e.g. as        further described below) and do not require the use of mammalian        expression systems, as with for example conventional antibodies;    -   immunoglobulin single variable domains are relatively small        (approximately 15 kDa, or 10 times smaller than a conventional        IgG) compared to conventional 4-chain antibodies and        antigen-binding fragments thereof, and therefore show high(er)        penetration into tissues (including but not limited to solid        tumors and other dense tissues) and can be administered in        higher doses than such conventional 4-chain antibodies and        antigen-binding fragments thereof;    -   VHHs have specific so-called “cavity-binding properties” (inter        alia due to their extended CDR3 loop, compared to VH domains        from 4-chain antibodies) and can therefore also access targets        and epitopes not accessible to conventional 4-chain antibodies        and antigen-binding fragments thereof;    -   VHHs have the particular advantage that they are highly soluble        and very stable and do not have a tendency to aggregate (as with        the mouse-derived antigen-binding domains described by Ward et        al., Nature 341: 544-546 (1989)).

The immunoglobulin single variable domains of the invention are notlimited with respect to a specific biological source from which theyhave been obtained or to a specific method of preparation. For example,obtaining VHHs may include the following steps:

(1) isolating the VHH domain of a naturally occurring heavy chainantibody; or screening a library comprising heavy chain antibodies orVHHs and isolating VHHs therefrom;

(2) expressing a nucleic acid molecule encoding a VHH with the naturallyoccurring sequence;

(3) “humanizing” (as described herein) a VHH, optionally after affinitymaturation, with a naturally occurring sequence or expressing a nucleicacid encoding such humanized VHH;

(4) “camelizing” (as described below) a immunoglobulin single variableheavy domain from a naturally occurring antibody from an animal species,in particular a species of mammal, such as from a human being, orexpressing a nucleic acid molecule encoding such camelized domain;

(5) “camelizing” a VH, or expressing a nucleic acid molecule encodingsuch a camelized VH;

(6) using techniques for preparing synthetically or semi-syntheticallyproteins, polypeptides or other amino acid sequences;

(7) preparing a nucleic acid molecule encoding a VHH domain usingtechniques for nucleic acid synthesis, followed by expression of thenucleic acid thus obtained;

(8) subjecting heavy chain antibodies or VHHs to affinity maturation, tomutagenesis (e.g. random mutagenesis or site-directed mutagenesis)and/or any other technique(s) in order to increase the affinity and/orspecificity of the VHH; and/or

(9) combinations or selections of the foregoing steps.

Suitable methods and techniques for performing the above-described stepsare known in the art and will be clear to the skilled person. By way ofexample, methods of obtaining VHH domains binding to a specific antigenor epitope have been described in WO2006/040153 and WO2006/122786.

According to specific embodiments, the immunoglobulin single variabledomains of the invention or present in the polypeptides of the inventionare VHH domains with an amino acid sequence that essentially correspondsto the amino acid sequence of a naturally occurring VHH domain, but thathas been “humanized” or “sequence-optimized” (optionally afteraffinity-maturation), i.e. by replacing one or more amino acid residuesin the amino acid sequence of said naturally occurring VHH sequence byone or more of the amino acid residues that occur at the correspondingposition(s) in a variable heavy domain of a conventional 4-chainantibody from a human being. This can be performed using methods knownin the art, which can by routinely used by the skilled person.

A humanized VHH domain may contain one or more fully human frameworkregion sequences, and, in an even more specific embodiment, may containhuman framework region sequences derived from the human germline Vh3sequences DP-29, DP-47, DP-51, or parts thereof, or be highly homologousthereto, optionally combined with JH sequences, such as JH5. Thus, ahumanization protocol may comprise the replacement of any of the VHHresidues with the corresponding framework 1, 2 and 3 (FR1, FR2 and FR3)residues of germline VH genes such as DP 47, DP 29 and DP 51) eitheralone or in combination. Suitable framework regions (FR) of theimmunoglobulin single variable domains of the invention can be selectedfrom those as set out e.g. in WO2006/004678 and specifically, includethe so-called “KERE” and “GLEW” classes. Examples are immunoglobulinsingle variable domains having the amino acid sequence G-L-E-W at aboutpositions 44 to 47, and their respective humanized counterparts. Ahumanized VHH domain may contain one or more fully human frameworkregion sequences.

By way of example, a humanizing substitution for VHHs belonging to the103 P,R,S-group and/or the GLEW-group (as defined below) is 108Q to108L. Methods for humanizing immunoglobulin single variable domains areknown in the art.

Binding immunoglobulin single variable domains with improved propertiesin view of therapeutic application, e.g. enhanced affinity or decreasedimmunogenicity, may be obtained from individual binding molecules bytechniques known in the art, such as affinity maturation (for example,starting from synthetic, random or naturally occurring immunoglobulinsequences), CDR grafting, humanizing, combining fragments derived fromdifferent immunoglobulin sequences, PCR assembly using overlappingprimers, and similar techniques for engineering immunoglobulin sequenceswell known to the skilled person; or any suitable combination of any ofthe foregoing, also termed “sequence optimization”, as described herein.Reference is, for example, made to standard handbooks, as well as to thefurther description and Examples.

If appropriate, a binding molecule with increased affinity may beobtained by affinity-maturation of another binding molecule, the latterrepresenting, with respect to the affinity-matured molecule, the“parent” binding molecule.

Methods of obtaining VHHs that bind to a specific antigen or epitopehave been described earlier, e.g. in WO2006/040153 and WO2006/122786. Asalso described therein in detail, VHH domains derived from camelids canbe “humanized” (also termed “sequence-optimized” herein,“sequence-optimizing” may, in addition to humanization, encompass anadditional modification of the sequence by one or more mutations thatfurnish the VHH with improved properties, such as the removal ofpotential post translational modification sites) by replacing one ormore amino acid residues in the amino acid sequence of the original VHHsequence by one or more of the amino acid residues that occur at thecorresponding position(s) in a VH domain from a conventional 4-chainantibody from a human being. A humanized VHH domain can contain one ormore fully human framework region sequences, and, in an even morespecific embodiment, can contain human framework region sequencesderived from DP-29, DP-47, DP-51, or parts thereof, optionally combinedwith JH sequences, such as JH5.

Domain antibodies, also known as “Dab”s and “dAbs” (the terms “DomainAntibodies” and “dAbs” being used as trademarks by the GlaxoSmithKlinegroup of companies) have been described in e.g. Ward, E. S., et al.:“Binding activities of a repertoire of single immunoglobulin variabledomains secreted from Escherichia coli”; Nature 341: 544-546 (1989);Holt, L. J. et al.: “Domain antibodies: proteins for therapy”; TRENDS inBiotechnology 21(11): 484-490 (2003); and WO2003/002609.

Domain antibodies essentially correspond to the VH or VL domains ofantibodies from non-camelid mammals, in particular human 4-chainantibodies. In order to bind an epitope as a single antigen bindingdomain, i.e. without being paired with a VL or VH domain, respectively,specific selection for such antigen binding properties is required, e.g.by using libraries of human single VH or VL domain sequences.

Domain antibodies have, like VHHs, a molecular weight of approximately13 to approximately 16 kDa and, if derived from fully human sequences,do not require humanization for e.g. therapeutical use in humans. As inthe case of VHH domains, they are well expressed also in prokaryoticexpression systems, providing a significant reduction in overallmanufacturing cost.

Furthermore, it will also be clear to the skilled person that it ispossible to “graft” one or more of the CDR's mentioned above onto other“scaffolds”, including but not limited to human scaffolds ornon-immunoglobulin scaffolds. Suitable scaffolds and techniques for suchCDR grafting are known in the art.

The terms “epitope” and “antigenic determinant”, which can be usedinterchangeably, refer to the part of a macromolecule, such as apolypeptide, that is recognized by antigen-binding molecules, such asconventional antibodies or the polypeptides of the invention, and moreparticularly by the antigen-binding site of said molecules. Epitopesdefine the minimum binding site for an immunoglobulin, and thusrepresent the target of specificity of an immunoglobulin.

A polypeptide (such as an immunoglobulin, an antibody, an immunoglobulinsingle variable domain of the invention, or generally an antigen-bindingmolecule or a fragment thereof) that can “bind to” or “specifically bindto”, that “has affinity for” and/or that “has specificity for” a certainepitope, antigen or protein (or for at least one part, fragment orepitope thereof) is said to be “against” or “directed against” saidepitope, antigen or protein or is a “binding” molecule with respect tosuch epitope, antigen or protein. In this context, a VEGF-bindingcomponent may also be referred to as “VEGF-neutralizing”.

Generally, the term “specificity” refers to the number of differenttypes of antigens or epitopes to which a particular antigen-bindingmolecule or antigen-binding protein (such as an immunoglobulin singlevariable domain of the invention) molecule can bind. The specificity ofan antigen-binding molecule can be determined based on its affinityand/or avidity. The affinity, represented by the equilibrium constantfor the dissociation of an antigen with an antigen-binding protein (KD),is a measure for the binding strength between an epitope and anantigen-binding site on the antigen-binding protein: the lesser thevalue of the KD, the stronger the binding strength between an epitopeand the antigen-binding molecule (alternatively, the affinity can alsobe expressed as the affinity constant (KA), which is 1/KD). As will beclear to the skilled person (for example on the basis of the furtherdisclosure herein), affinity can be determined in a manner known per se,depending on the specific antigen of interest. Avidity is the measure ofthe strength of binding between an antigen-binding molecule (such as animmunoglobulin, an antibody, an immunoglobulin single variable domain ora polypeptides containing it and the pertinent antigen. Avidity isrelated to both the affinity between an epitope and its antigen bindingsite on the antigen-binding molecule and the number of pertinent bindingsites present on the antigen-binding molecule.

The part of an antigen-binding molecule that recognizes the epitope iscalled a paratope.

Unless indicated otherwise, the term “VEGF-binding molecule” or“Ang2-binding molecule” includes anti-VEGF or anti-Ang2 antibodies,anti-VEGF antibody or anti-Ang2 antibody fragments, “anti-VEGFantibody-like molecules” or “anti-Ang2 antibody-like molecules”, asdefined herein, and conjugates with any of these. Antibodies include,but are not limited to, monoclonal and chimerized monoclonal antibodies.The term “antibody” encompasses complete immunoglobulins, likemonoclonal antibodies produced by recombinant expression in host cells,as well as antibody fragments or “antibody-like molecules”, includingsingle-chain antibodies and linear antibodies, so-called “SMIPs” (“SmallModular Immunopharmaceuticals”), as e.g described in WO2002/056910;Antibody-like molecules include immunoglobulin single variable domains,as defined herein. Other examples for antibody-like molecules areimmunoglobulin super family antibodies (IgSF), or CDR-grafted molecules.

“Ang2-binding molecule” or “VEGF-binding molecule” respectively, refersto both monovalent target-binding molecules (i.e. molecules that bind toone epitope of the respective target) as well as to bi- or multivalentbinding molecules (i.e. binding molecules that bind to more than oneepitope, e.g. “biparatopic” molecules as defined hereinbelow). Ang2 (orVEGF)-binding molecules containing more than one Ang2 (or VEGF)-bindingimmunoglobulin single variable domain are also termed “formatted”binding molecules, they may, within the target-binding component, inaddition to the immunoglobulin single variable domains, comprise linkersand/or moieties with effector functions, e.g. half-life-extendingmoieties like albumin-binding immunoglobulin single variable domains,and/or a fusion partner like serum albumin and/or an attached polymerlike PEG.

The term “biparatopic Ang2 (or VEGF)-binding molecule” or “biparatopicimmunoglobulin single variable domain” as used herein shall mean abinding molecule comprising a first immunoglobulin single variabledomain and a second immunoglobulin single variable domain as hereindefined, wherein the two molecules bind to two non-overlapping epitopesof the respective antigen. The biparatopic binding molecules arecomposed of immunoglobulin single variable domains which have differentspecificities with respect to the epitope. The part of anantigen-binding molecule (such as an antibody or an immunoglobulinsingle variable domain of the invention) that recognizes the epitope iscalled a paratope.

A formatted binding molecule may, albeit less preferred, also comprisetwo identical immunoglobulin single variable domains or two differentimmunoglobulin single variable domains that recognize the same oroverlapping epitopes or their respective antigen. In this case, withrespect to VEGF, the two immunoglobulin single variable domains may bindto the same or an overlapping epitope in each of the two monomers thatform the VEGF dimer.

Typically, the binding molecules of the invention will bind with adissociation constant (K_(D)) of 10 E-5 to 10 E-14 moles/liter (M) orless, and preferably 10 E-7 to 10 E-14 moles/liter (M) or less, morepreferably 10 E-8 to 10 E-14 moles/liter, and even more preferably 10E-11 to 10 E-13, as measured e.g. in a Biacore or in a Kinexa assay),and/or with an association constant (K_(A)) of at least 10 E7 ME-1,preferably at least 10 E8 ME-1, more preferably at least 10 E9 ME-1,such as at least 10 E11 ME-1. Any K_(D) value greater than 10 E-4 M isgenerally considered to indicate non-specific binding.

Preferably, a polypeptide of the invention will bind to the desiredantigen, i.e. VEGF or Ang2, respectively, with a K_(D) less than 500 nM,preferably less than 200 nM, more preferably less than 10 nM, such asless than 500 pM. Specific binding of an antigen-binding protein to anantigen or epitope can be determined in any suitable manner known perse, including, for example, the assays described herein, Scatchardanalysis and/or competitive binding assays, such as radioimmunoassays(RIA), enzyme immunoassays (EIA) and sandwich competition assays, andthe different variants thereof known per se in the art.

Amino acid residues will be indicated according to the standardthree-letter or one-letter amino acid code, as generally known andagreed upon in the art. When comparing two amino acid sequences, theterm “amino acid difference” refers to insertions, deletions orsubstitutions of the indicated number of amino acid residues at aposition of the reference sequence, compared to a second sequence. Incase of substitution(s), such substitution(s) will preferably beconservative amino acid substitution(s), which means that an amino acidresidue is replaced with another amino acid residue of similar chemicalstructure and which has little or essentially no influence on thefunction, activity or other biological properties of the polypeptide.Such conservative amino acid substitutions are well known in the art,for example from WO1998/49185, wherein conservative amino acidsubstitutions preferably are substitutions in which one amino acidwithin the following groups (i)-(v) is substituted by another amino acidresidue within the same group: (i) small aliphatic, nonpolar or slightlypolar residues: Ala, Ser, Thr, Pro and Gly; (ii) polar, negativelycharged residues and their (uncharged) amides: Asp, Asn, Glu and Gin;(iii) polar, positively charged residues: H is, Arg and Lys; (iv) largealiphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and (v)aromatic residues: Phe, Tyr and Trp. Particularly preferred conservativeamino acid substitutions are as follows: Ala into Gly or into Ser; Arginto Lys; Asn into Gin or into H is; Asp into Glu; Cys into Ser; Gininto Asn; Glu into Asp; Gly into Ala or into Pro; H is into Asn or intoGin; Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg,into Gin or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met,into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr intoTrp or into Phe; Val into Ile or into Leu.

A polypeptide or nucleic acid molecule is considered to be “(in)essentially isolated (form)”—for example, when compared to its nativebiological source and/or the reaction medium or cultivation medium fromwhich it has been obtained—when it has been separated from at least oneother component with which it is usually associated in said source ormedium, such as another protein/polypeptide, another nucleic acid,another biological component or macromolecule or at least onecontaminant, impurity or minor component. In particular, a polypeptideor nucleic acid molecule is considered “essentially isolated” when ithas been purified at least 2-fold, in particular at least 10-fold, morein particular at least 100-fold, and up to 1000-fold or more. Apolypeptide or nucleic acid molecule that is “in essentially isolatedform” is preferably essentially homogeneous, as determined using asuitable technique, such as a suitable chromatographical technique, suchas polyacrylamide gel electrophoresis.

“Sequence identity” between two VEGF-binding molecule sequences orbetween two Ang2-binding molecule sequences indicates the percentage ofamino acids that are identical between the sequences. It may becalculated or determined as described in paragraph f) on pages 49 and 50of WO2008/020079. “Sequence similarity” indicates the percentage ofamino acids that either are identical or that represent conservativeamino acid substitutions.

Alternative methods for numbering the amino acid residues of V_(H)domains, which methods can also be applied in an analogous manner to VHHdomains, are known in the art. However, in the present description,claims and figures, the numbering according to Kabat and applied to VHHdomains as described above will be followed, unless indicated otherwise.

An “affinity-matured” VEGF-binding molecule or Ang2-binding molecule, inparticular a VHH or a domain antibody, has one or more alterations inone or more CDRs which result in an improved affinity for VEGF or Ang2,as compared to the respective parent VEGF-binding molecule orAng2-binding molecule. Affinity-matured VEGF-binding molecules orAng2-binding molecules of the invention may be prepared by methods knownin the art, for example, as described by Marks et al., 1992,Biotechnology 10: 779-783, or Barbas, et al., 1994, Proc. Nat. Acad.Sci, USA 91: 3809-3813.; Shier et al., 1995, Gene 169:147-155; Yelton etal., 1995, Immunol. 155: 1994-2004; Jackson et al., 1995, J. Immunol.154(7):3310-9; and Hawkins et al., 1992, J. Mol. Biol. 226(3): 889 896;KS Johnson and RE Hawkins, “Affinity maturation of antibodies usingphage display”, Oxford University Press 1996.

For the present invention, an “amino acid sequences of SEQ ID NO: x”:includes, if not otherwise stated, an amino acid sequence that is 100%identical with the sequence shown in the respective SEQ ID NO: x;

-   -   a) amino acid sequences that have at least 80% amino acid        identity with the sequence shown in the respective SEQ ID NO: x;    -   b) amino acid sequences that have 3, 2, or 1 amino acid        differences with the sequence shown in the respective SEQ ID NO:        x.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer to be treatedwith a bispecific binding molecule of the invention, include but are notlimited to carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers, as suggested for treatment withVEGF antagonists in US 2008/0014196, include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidneycancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, gastric cancer, melanoma, and various types of headand neck cancer. Dysregulation of angiogenesis can lead to manydisorders that can be treated by compositions and methods of theinvention. These disorders include both non-neoplastic and neoplasticconditions. Neoplasties include but are not limited those describedabove.

Non-neoplastic disorders include, but are not limited to, as suggestedfor treatment with VEGF antagonists in US2008/0014196, undesired oraberrant hypertrophy, arthritis, rheumatoid arthritis (RA), psoriasis,psoriatic plaques, sarcoidosis, atherosclerosis, atheroscleroticplaques, diabetic and other proliferative retinopathies includingretinopathy of prematurity, retrolental fibroplasia, neovascularglaucoma, age-related macular degeneration, diabetic macular edema,corneal neovascularization, corneal graft neovascularization, cornealgraft rejection, retinal/choroidal neovascularization,neovascularization of the angle (rubeosis), ocular neovascular disease,vascular restenosis, arteriovenous malformations (AVM), meningioma,hemangioma, angiofibroma, thyroid hyperplasias (including Grave'sdisease), corneal and other tissue transplantation, chronicinflammation, lung inflammation, acute lung injury/ARDS, sepsis, primarypulmonary hypertension, malignant pulmonary effusions, cerebral edema(e.g., associated with acute stroke/closed head injury/trauma), synovialinflammation, pannus formation in RA, myositis ossificans, hypertropicbone formation, osteoarthritis (OA), refractory ascites, polycysticovarian disease, endometriosis, 3^(rd) spacing of fluid diseases(pancreatitis, compartment syndrome, burns, bowel disease), uterinefibroids, premature labor, chronic inflammation such as IBD (Crohn'sdisease and ulcerative colitis), renal allograft rejection, inflammatorybowel disease, nephrotic syndrome, undesired or aberrant tissue massgrowth (non-cancer), hemophilic joints, hypertrophic scars, inhibitionof hair growth, Osier-Weber syndrome, pyogenic granuloma retrolentalfibroplasias, scleroderma, trachoma, vascular adhesions, synovitis,dermatitis, preeclampsia, ascites, pericardial effusion (such as thatassociated with pericarditis), and pleural effusion.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention relates to a bispecific bindingmolecule comprising at least one Ang2-binding component and at least oneVEGF-binding component.

In a preferred embodiment, the present invention relates to a bispecificbinding molecule comprising at least one VEGF-binding component and atleast one Ang2-binding component which further comprises at least afurther binding component, preferably a serum albumin binding component(serum albumin binding molecule).

In a preferred embodiment, the serum albumin binding component of thebinding molecule of the present invention is an isolated immunoglobulinsingle variable domain or a polypeptide containing one or more of saidimmunoglobulin single variable domains, wherein said immunoglobulinsingle variable domain consists of four framework regions and threecomplementarity determining regions CDR1, CDR2 and CDR3, respectively,and wherein said CDR3 has an amino acid sequence selected from aminoacid sequences shown in SEQ ID NOs: 257, 260, 263, 266, 269, 272, or275.

More preferably, said one or more immunoglobulin single variable domainof the serum albumin binding component contain

-   a. a CDR3 with an amino acid sequence selected from a first group of    amino acid sequences shown in SEQ ID NOs: SEQ IDs NOs: 257, 260,    263, 266, 269, 272, or 275;-   b. a CDR1 with an amino acid sequences selected from a second group    of amino acid sequences shown SEQ ID NOs:255, 258, 261, 264, 267,    270, or 273;-   c. a CDR2 with an amino acid sequences selected from a second group    of amino acid sequences shown SEQ ID NOs:256, 259, 262, 265, 268,    271, or 274.

In a more preferred embodiment, said one or more immunoglobulin singlevariable domains of the serum albumin binding component are VHHs,preferably having an amino acid sequence shown in SEQ ID NOs: 98 or 254.

According to preferred embodiments, said Ang2-binding component and saidVEGF-binding component comprise at least one Ang2-binding immunoglobulinsingle variable domain and at least one VEGF-binding immunoglobulinsingle variable domain, respectively.

In a preferred aspect, said Ang2-binding component and said VEGF-bindingcomponent each comprise at least one VEGF-binding immunoglobulin singlevariable domain and at least one Ang2-binding immunoglobulin singlevariable domain, respectively, wherein each of said immunoglobulinsingle variable domains has four framework regions and threecomplementarity determining regions CDR1, CDR2 and CDR3, respectively.

Thus, the anti-Ang2 and/or the anti-VEGF component contained in thebispecific binding molecules of the invention may include two (or more)anti-Ang2 (or anti-VEGF, respectively) immunoglobulin single variabledomains, wherein the immunoglobulin single variable domains are directedagainst different epitopes within the Ang2 (or VEGF) target. Thus, thetwo immunoglobulin single variable domains in a bispecific bindingmolecule will have different antigen specificity and therefore differentCDR sequences.

Such bivalent binding molecules are also named “biparatopic singledomain antibody constructs” (if the immunoglobulin single variabledomains consist or essentially consist of single domain antibodies), or“biparatopic VHH constructs” (if the immunoglobulin single variabledomains consist or essentially consist of VHHs), respectively, as thetwo immunoglobulin single variable domains will include two differentparatopes.

In the bispecific binding molecule of the invention, one or both of thebinding molecules may be bivalent; e.g. the VEGF-binding component maybe biparatopic and the Ang2-binding component may be one immunoglobulinsingle variable domain, or the VEGF-binding component may be oneimmunoglobulin single variable domain and the Ang2 is binding componentmay be biparatopic.

In bispecific binding molecules of the invention, it is preferably theVEGF-binding component that contains a bivalent VEGF-bindingimmunoglobulin single variable domain, e.g. a biparatopic VHH.

Such VEGF-binding immunoglobulin single variable domain may be two ormore VEGF-binding VHHs, which are

-   a. identical VHHs that are capable of blocking the interaction    between recombinant human VEGF and the recombinant human VEGFR-2    with an inhibition rate of a 60% or-   b. different VHHs that bind to non-overlapping epitopes of VEGF,    wherein at least one VHH is capable of blocking the interaction    between recombinant human VEGF and the recombinant human VEGFR-2    with an inhibition rate of ≧60% and wherein at least one VHH is    capable of blocking said interaction with an inhibition rate of s    60%.

The VEGF-binding component comprising at least a variable domain withfour framework regions and three complementarity determining regionsCDR1, CDR2 and CDR3, respectively, wherein said CDR3 has the amino acidsequence Ser Arg Ala Tyr Xaa Ser Xaa Arg Leu Arg Leu Xaa Xaa Thr Tyr XaaTyr as shown in SEQ ID NO: 1,

wherein

Xaa at position 5 is Gly or Ala;

Xaa at position 7 is Ser or Gly;

Xaa at position 12 is Gly, Ala or Pro;

Xaa at position 13 is Asp or Gly;

Xaa at position 16 is Asp or Glu; and

wherein said VEGF-binding component is capable of blocking theinteraction of human recombinant VEGF165 with the human recombinantVEGFR-2 with an inhibition rate of ≧60%.

According to preferred embodiments, Xaa at position 5 is Gly, Xaa atposition 7 is Ser, Xaa at position 12 is Ala, and Xaa at position 13 isAsp.

In particular, said CDR3 has a sequence selected from

SEQ ID NO: 2 SRAYGSSRLRLGDTYDY, SEQ ID NO: 3 SRAYGSSRLRLADTYDY;SEQ ID NO: 4 SRAYGSSRLRLADTYEY; SEQ ID NO: 5 SRAYGSGRLRLADTYDY;SEQ ID NO: 6 SRAYASSRLRLADTYDY; SEQ ID NO: 7 SRAYGSSRLRLPDTYDY;SEQ ID NO: 8 SRAYGSSRLRLPGTYDY.

According to certain embodiments, a VEGF-binding component comprises oneor more immunoglobulin single variable domains each containing

-   -   a. a CDR3 with an amino acid sequence selected from a first        group of sequences shown in SEQ ID NO: 2 to 8;    -   b. a CDR1 and a CDR2 with an amino acid sequences that is        contained, as indicated in Table 3, in a sequence selected from        a second group of amino acid sequences shown in SEQ ID NOs: 9 to        46, wherein said second sequence contains the respective CDR3        selected according to a).

According to preferred embodiments, the immunoglobulin single variabledomains are VHHs.

According to specific embodiments, the VHHs have amino acid sequencesselected from sequences shown in SEQ ID NOs: 9-46.

According to another specific embodiment, the VHHs have amino acidsequences selected from SEQ ID NOs: 15, SEQ ID NO: 18 and SEQ ID NO: 25.

The invention also relates to VEGF-binding component that have beenobtained by affinity maturation and/or sequence optimization of anabove-defined VHH, e.g. to a VHH that has been obtained by sequenceoptimization of a VHH having an amino acid sequence shown in SEQ ID NO:18. Examples are VHHs having amino acid sequences selected fromsequences shown in SEQ ID NOs: 47-57.

According to certain embodiments, a VEGF-binding domain of the inventionmay be formatted, as herein defined, e.g. it may be biparatopic orcomprise two identical immunoglobulin single variable domains. SuchVEGF-binding components may comprise two or more VHHs, which are

-   -   a) identical VHHs that are capable of blocking the interaction        between recombinant human VEGF and the recombinant human VEGFR-2        with an inhibition rate of ≧60% or    -   b) different VHHs that bind to non-overlapping epitopes of VEGF,        wherein at least one VHH is capable of blocking the interaction        between recombinant human VEGF and the recombinant human VEGFR-2        with an inhibition rate of ≧60% and wherein at least one VHH        binds is capable of blocking said interaction with an inhibition        rate of ≧60%.

The percentage of blocking said interaction at an inhibition rate of≧60% or ≧60%, respectively, refers to an inhibition rate as determinedby an Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen®),a competition ELISA, a plasmon resonance (SPR) based assay (Biacore®) asused in the Examples.

In the following, the ability of VHHs according to a) is also termed“receptor-blocking”, while the ability of VHHs according to b) is alsotermed “non-receptor-blocking”.

Preferably, the receptor-blocking VHHs have an inhibition rate of ≧80%,more preferably ≧90%; the most preferred VHHs being complete receptorblockers, i.e. have an inhibition rate of 100%.

A VEGF-binding component may contain two or more identical VHHs a)selected from VHHs having amino acid sequences shown in SEQ ID NOs: 9-46or VHHs that have been obtained by affinity maturation and/or sequenceoptimization of such VHH. The VHH may be selected from VHHs having theamino acid shown in SEQ ID NO: 18 or SEQ ID NO: 47-57.

According to preferred embodiments, a formatted VEGF-binding componentcomprises two VHHs each having the amino acid sequence shown in SEQ IDNO: 57.

In formatted VEGF-binding components comprising two different VHHs

-   -   a) said one or more VHHs with an inhibition rate of ≧60% are        selected from        -   i. VHHs having an amino acid sequence selected from amino            acid sequences shown in SEQ ID NOs: 9-46 or        -   ii. VHHs that have been obtained by affinity maturation            and/or sequence optimization of such VHHs, and wherein    -   b) said one or more VHHs with an inhibition rate of ≧60% are        selected from        -   i. SEQ ID NOs: 58-124 or        -   ii. VHHs that have been obtained by affinity maturation            and/or sequence optimization of such VHH.

According to preferred embodiments, two VHHs are contained inpolypeptides with amino acid sequences shown in SEQ ID NOs: 128-168,separated by linker sequences as indicated in Table 15.

In a preferred VEGF-binding component VHH a) i. has an amino acidsequence shown in SEQ ID NO: 18 and VHH b) i. has an amino acid sequenceshown in SEQ ID NO: 64.

In other preferred VEGF-binding components VHHs according to a) ii. areselected from VHHs having an amino acid sequence shown in SEQ ID NOs:47-57 and VHHs according to b) ii. are selected from VHHs having anamino acid sequence shown in SEQ ID NOs: 125-127.

Particularly preferred is a biparatopic VEGF-binding componentcomprising two VHHs, one of them having the amino acid shown in SEQ IDNO: 57 and one of them having the amino acid shown in SEQ ID NO: 127.

The Ang2-binding component comprises at least a variable domain withfour framework regions and three complementarity determining regionsCDR1, CDR2 and CDR3, respectively, wherein said CDR3 has an amino acidsequence selected from amino acid sequences shown in SEQ ID NOs: 226,229, 232, 235, 238, 241, 244, 247, 250, or 253.

In a second aspect, said Ang2-binding component is an isolatedimmunoglobulin single variable domain or a polypeptide containing one ormore of said immunoglobulin single variable domains, wherein saidimmunoglobulin single variable domain consists of four framework regionsand three complementarity determining regions CDR1, CDR2 and CDR3,respectively, and wherein said CDR3 has an amino acid sequence selectedfrom amino acid sequences shown in SEQ ID NOs: 226, 229, 232, 235, 238,241, 244, 247, 250, or 253.

In a further aspect, said immunoglobulin single variable domain of theAng2-binding component contains

-   a. a CDR3 with an amino acid sequence selected from a first group of    amino acid sequences shown in SEQ ID NOs: SEQ IDs NOs: 226, 229,    232, 235, 238, 241, 244, 247, 250, or 253 (see also Table 49);-   b. a CDR1 with an amino acid sequences that is contained, as    indicated in Table 36-A, 38-A, 41-A, or 45-A, as partial sequence in    a sequence selected from a second group of amino acid sequences    shown SEQ ID NOs: 224, 227, 230, 233, 236, 239, 242, 245, 248, or    251 (see also Table 49);-   c. a CDR2 with an amino acid sequences that is contained, as    indicated in Table 36-A, 38-A, 41-A, or 45-A, as partial sequence in    a sequence selected from a second group of amino acid sequences    shown SEQ ID NOs: 225, 228, 231, 234, 237, 240, 243, 246, 249, or    252 (see also Table 49).

Preferably, the immunoglobulin single variable domain of theAng2-binding component is a VHH, preferably having amino acid sequenceselected from amino acid sequences shown in SEQ ID NOs: 214, 215, 216,217, 218, 219, 220, 221, 222, or 223.

In another preferred embodiment, the immunoglobulin single variabledomain of the Ang2-binding component has been obtained by affinitymaturation or humanization of an immunoglobulin single variable domainas described herein.

Similarly, the present invention also relates to a VHH which has beenobtained by affinity maturation or humanization of a VHH of theAng2-binding component as described herein.

The present invention thus also relates to an Ang2-binding VHH with anamino acid sequence selected from acid sequences shown in SEQ ID NOs:214, 215, 216, 217, 218, 219, 220, 221, 222, or 223.

Suitable parent Ang2-binding components for affinity maturation are, byway of example, the above-described VHHs with amino acid sequences shownin SEQ ID NOs:214, 215, 216, 217, 21i8, or 219.

Accordingly, the invention also relates to Ang2-binding molecules thathave been obtained by affinity maturation and/or sequence optimizationof an above-defined VHH, e.g. to a VHH that has been obtained bysequence optimization of a VHH having an amino acid sequence shown asSEQ ID NOs: 217, 218, 219, 220, 221, 222, or 223. The “source” aminoacid sequences that were used to generate the latter VHHs are shown inSEQ ID NOs: 214, 215, or 216. Also these amino acid sequences aresuitable Ang2-binding components that can be applied in the bindingmolecules of the present invention.

As described herein, the binding molecule of the present inventionpreferably comprises at least one serum albumin binding component.Particularly preferred binding molecules thus have at least oneVEGF-binding component, at least one Ang2-binding component and at leastone serum albumin binding component. The order of these three bindingcomponents could be any possible order such as the order set out inTable 36-B, 38-B, 40, 41-B, 42, 43, 45-B, 46-A, or 47-A; or in FIG. 20,23, 27, or 30, e.g. the VEGF-, Ang2- or serum albumin binding componentcan be N-terminal or C-terminal. Notably, “1D01” (SEQ ID No: 214),“11B07”, “00027” (SEQ ID No:216), “00908”, “7G08” (SEQ ID No:215),“00919”, “00921” (SEQ ID No: 220), “00928” (SEQ ID No:221), “00932”,“00933”, “00934”, “00935”, “00936”, “00937”, “00938” (SEQ ID No:222), or“00956” (SEQ is ID No:223) as referred to in the legend of theaforementioned Tables and Figures stand for Ang2-binding components,while “00038” stands for a VEGF-binding component and “ALB11” stands fora serum albumin binding component. None of them is to be construed to aspecific sequence, but stands for a Ang2-, VEGF- and serum albuminbinding component in general when used in the context of possibleset-ups of binding molecules of the present invention.

However, it is preferred that the serum albumin binding component is inbetween the VEGF- and Ang2-binding component (or vice versa), while itis particularly preferred that at least one VEGF-binding component isN-terminal, followed by at least one serum albumin binding component,followed by at least one Ang2-binding component at the C-Terminus. Thisset-up is shown to be specifically useful.

The present invention relates thus in a preferred aspect to bindingmolecules comprising at least one VEGF-binding component, at least oneAng2-binding component and at least one serum albumin binding componenthaving an amino acid sequence selected from the amino acid sequencesshown in SEQ ID NOs: 180-213,

“At least one” binding component (VEGF, Ang2 or serum albumin) when usedherein includes that a binding molecule of the present invention maycontain one, two, three, four or five VEGF-, Ang2-, and/or serum albuminbinding components (i.e., entities/units) which are preferablyrepresented by an immunoglobulin singly variable domain as describedherein.

The VEGF- and/or Ang2-binding components with improved properties inview of therapeutic application, e.g. enhanced affinity or decreasedimmunogenicity, may be obtained from individual VEGF- or Ang2-bindingcomponents of the invention by techniques known in the art, such asaffinity maturation (for example, starting from synthetic, random ornaturally occurring immunoglobulin sequences), CDR grafting, humanizing,combining fragments derived from different immunoglobulin sequences, PCRassembly using overlapping primers, and similar techniques forengineering immunoglobulin sequences well known to the skilled person;or any suitable combination of any of the foregoing, also termed“sequence optimization”, as described herein. Reference is, for example,made to standard handbooks, as well as to the further description andExamples.

If appropriate, a VEGF- or Ang2-binding component of the invention withincreased affinity may be obtained by affinity-maturation of anotherVEGF- or Ang2-binding component, the latter representing, with respectto the affinity-matured molecule, the “parent” VEGF-binding component.

In VEGF or Ang2 VHHs of the invention that start with EVQ, theN-terminal E may be replaced by a D (which is often a result ofsequence-optimization) or it may be missing (as for expression of theVHH in E. coli). For formatted VEGF-binding components, this usuallyapplies only to the VHH that is situated N-terminally.

A preferred, but non-limiting humanizing substitution for VEGF VHHdomains belonging to the 103 P,R,S-group and/or the GLEW-group (asdefined below) is 108Q to 108L. Methods for humanizing immunoglobulinsingle variable domains are known in the art.

According to another embodiment, the immunoglobulin single variabledomain is a domain antibody, as defined herein.

In yet another embodiment, the representatives of the class of VEGF-and/or Ang2-binding immunoglobulin single variable domains of theinvention have amino acid sequences that correspond to the amino acidsequence of a naturally occurring VH domain that has been “camelized”,i.e. by replacing one or more amino acid residues in the amino acidsequence of a naturally occurring variable heavy chain from aconventional 4-chain antibody by one or more amino acid residues thatoccur at the corresponding position(s) in a VHH domain of a heavy chainantibody. This can be performed in a manner known per se, which will beclear to the skilled person, and reference is additionally be made toWO1994/04678. Such camelization may preferentially occur at amino acidpositions which are present at the VH-VL interface and at the so-calledCamelidae Hallmark residues (see for example also WO1994/04678). Adetailed description of such “humanization” and “camelization”techniques and preferred framework region sequences consistent therewithcan additionally be taken from e.g. pp. 46 and pp. 98 of WO2006/040153and pp. 107 of WO2006/122786.

The VEGF-binding components of the invention, e.g. immunoglobulin,single variable domains, have specificity for VEGF in that they compriseone or more immunoglobulin single variable domains specifically bindingto one or more epitopes within the VEGF molecule. The same is true forAng2-binding components of the invention.

Specific binding of an VEGF-binding component to its antigen VEGF can bedetermined in any suitable manner known per se, including, for example,the assays described herein, Scatchard analysis and/or competitivebinding assays, such as radioimmunoassays (RIA), enzyme immunoassays(EIA and ELISA) and sandwich competition assays, and the differentvariants thereof known per se in the art. The same is true for anAng2-binding component when binding to its antigen.

With regard to the antigen VEGF, a VEGF-binding component of theinvention, e.g. an immunoglobulin single variable domain, is not limitedwith regard to the species. Thus, the immunoglobulin single variabledomains of the invention preferably bind to human VEGF, if intended fortherapeutic purposes in humans. However, immunoglobulin single variabledomains that bind to VEGF from another mammalian species are also withinthe scope of the invention. An immunoglobulin single variable domain ofthe invention binding to one species form of VEGF may cross-react withVEGF, which has a different sequence than the human one, from one ormore other species. For example, immunoglobulin single variable domainsof the invention binding to human VEGF may exhibit cross reactivity withVEGF from one or more other species of primates and/or with VEGF fromone or more species of animals that are used in animal models fordiseases, for example monkey, mouse, rat, rabbit, pig, dog, and inparticular in animal models for diseases and disorders associated withVEGF-mediated effects on angiogenesis (such as the species and animalmodels mentioned herein). Immunoglobulin single variable domains of theinvention that show such cross-reactivity are advantageous in a researchand/or drug development, since it allows the immunoglobulin singlevariable domains of the invention to be tested in acknowledged diseasemodels such as monkeys, in particular Cynomolgus or Rhesus, or mice andrats.

Preferably, in view of cross-reactivity with one or more VEGF moleculesfrom species other than human that is/are intended for use as an animalmodel during development of a therapeutic VEGF antagonist, aVEGF-binding component recognizes an epitope in a region of the VEGF ofinterest that has a high degree of identity with human VEGF.

An immunoglobulin single variable domain of the invention recognizes anepitope which is, totally or in part, located in a region of VEGF thatis relevant for binding to its receptor, in particular to VEGFR-2, whichhas been shown to be the receptor whose activation is causally involvedin the neovascularisation of tumors. According to preferred aspects,immunoglobulin single variable domains of the invention block VEGFreceptor activation, in particular VEGFR-2 activation, at leastpartially, preferably substantially and most preferably totally.

As described above, the ability of a VEGF-binding component to block theinteraction between VEGF and its receptors, in particular the VEGFR-2,can be determined by an Amplified Luminescent Proximity HomogeneousAssay (AlphaScreen®), a competition ELISA, or a plasmon resonance (SPR)based assay (Biacore®), as described in the Examples.

Preferably, an immunoglobulin single variable domain of the inventionbinds to VEGF with an affinity less than 500 nM, preferably less than200 nM, more preferably less than nM, such as less than 500 pM (asdetermined by Surface Plasmon Resonance analysis, as described inExample 5.7). The same is true for an immunoglobulin single variabledomain of the invention binds to angiopoietin.

Preferably, the immunoglobulin single variable domains of the inventionhave IC₅₀ values, as measured in a competition ELISA assay as describedin Example 5.1. in the range of 10⁻⁶ to 10.10 moles/litre or less, morepreferably in the range of 10⁻⁸ to 10⁻¹⁰ moles/litre or less and evenmore preferably in the range of 10⁻⁹ to 10⁻¹⁰ moles/litre or less.

According to a non-limiting but preferred embodiment of the invention,VEGF-binding immunoglobulin single variable domains of the inventionbind to VEGF with an dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹²moles/liter (M) or less, and preferably 10⁻⁷ to 10⁻¹² moles/liter (M) orless and more preferably 10⁻⁸ to 10⁻¹² moles/liter (M), and/or with anassociation constant (K_(A)) of at least 10⁷ M⁻¹, preferably at least10⁸ M⁻¹, more preferably at least 10⁹ M⁻¹, such as at least 10¹² M⁻¹;and in particular with a K_(D) less than 500 nM, preferably less than200 nM, more preferably less than 10 nM, such as less than 500 pM. TheK_(D) and K_(A) values of the immunoglobulin single variable domain ofthe invention against VEGF can be determined. The same is true for anAng2-binding immunoglobulin single variable domain of the invention.

Biparatopic VEGF-binding components comprising two or moreimmunoglobulin single variable domains essentially consist of orcomprise (i) a first immunoglobulin single variable domain specificallybinding to a first epitope of VEGF and (ii) a second immunoglobulinsingle variable domain specifically binding to a second epitope of VEGF,wherein the first epitope of VEGF and the second epitope of VEGF are notidentical epitopes. In other words, such polypeptide of the inventioncomprises or essentially consist of two or more immunoglobulin singlevariable domains that are directed against at least two non-overlappingepitopes present in VEGF, wherein said immunoglobulin single variabledomains are linked to each other in such a way that they are capable ofsimultaneously binding VEGF. In this sense, the polypeptide of theinvention can also be regarded as a “bivalent” or “multivalent”immunoglobulin construct, and especially as a “multivalentimmunoglobulin single variable domain construct”, in that thepolypeptide contains at least two binding sites for VEGF. (Suchconstructs are also termed “formatted” VEGF binding molecules, e.g.“formatted” VHHs). The same is true for biparatopic Ang2-bindingcomponents, mutatis mutandis.

Such VEGF- or Ang2-binding component of the invention includes (atleast) two anti-VEGF or Ang2 immunoglobulin single variable domains,respectively, wherein (the) two immunoglobulin single variable domainsare preferably directed against non-overlapping epitopes within the VEGFmolecule or angiopoietin molecule, respectively. Thus, these twoimmunoglobulin single variable domains will have a different antigenspecificity and therefore different CDR sequences. For this reason, suchpolypeptides of the invention will herein also be named “biparatopicpolypeptides”, or “biparatopic domain antibody constructs” (if theimmunoglobulin single variable domains consist or essentially consist ofdomain antibodies), or “biparatopic VHH constructs” (if theimmunoglobulin single variable domains consist or essentially consist ofVHHs), respectively, as the two immunoglobulin single variable domainswill include two different paratopes.

If a polypeptide of the invention is a biparatopic molecule as definedherein, at least one of the immunoglobulin single variable domaincomponents binds to an epitope such that the interaction betweenrecombinant human VEGF and recombinant human VEGFR-2 is blocked at aninhibition rate of ≧80%. As has been shown in experiments of theinvention, certain formatted molecules contain two VHHs that both blockthe VEGFR2 receptor at an inhibition rate of 280%. Certain VHHs of theinvention block the VEGFR-2 at an inhibition rate of 100%, i.e. they arecomplete blockers.

In both cases, additional sequences and moieties may be present withinthe VEGF-binding components of the invention, e.g. N-terminally,C-terminally, or located between the two immunoglobulin single variabledomains, e.g. linker sequences and sequences providing for effectorfunctions, as set out in more detail herein.

According to another, albeit less preferred embodiment, a VEGF-bindingcomponent of the invention may include more than two anti-VEGFimmunoglobulin single variable domains, i.e. three, four or even moreanti-VEGF VHHs. In this case, at least two of the anti-VEGFimmunoglobulin single variable domains are directed againstnon-overlapping epitopes within the VEGF molecule, wherein any furtherimmunoglobulin single variable domain may bind to any of the twonon-overlapping epitopes and/or a further epitope present in the VEGFmolecule.

According to the invention, the two or more immunoglobulin singlevariable domains can be, independently of each other, VHHs or domainantibodies, and/or any other sort of immunoglobulin single variabledomains, such as VL domains, as defined herein, provided that theseimmunoglobulin single variable domains will bind the antigen, i.e. VEGFor angiopoietin, respectively.

The detailed description of the binding components is primarily providedfor the VEGF-binding component. However, all features and optionsoutlined herein for the VEGF-binding component also apply equivalentlyfor the Ang2-binding component, mutatis mutandis.

According to preferred embodiments, the binding molecules present in thebispecific binding molecules (the Ang2-binding molecules within theAng2-binding component or the VEGF-binding molecules within theVEGF-binding component or the two adjacent Ang2- and VEGF-bindingcomponents) may be connected with each other directly (i.e. without useof a linker) or via a linker. The linker is preferably a linker peptideand will be selected so as to allow binding of the two different bindingmolecules to each of non-overlapping epitopes of the targets, eitherwithin one and the same target molecule, or within two differentmolecules.

In the case of biparatopic binding molecules, selection of linkerswithin the Ang2- or the VEGF-binding component will inter alia depend onthe epitopes and, specifically, the distance between the epitopes on thetarget to which the immunoglobulin single variable domains bind, andwill be clear to the skilled person based on the disclosure herein,optionally after some limited degree of routine experimentation.

Two binding molecules (two VHHs or domain antibodies or VHH and a domainantibody), or two binding components, may be linked to each other via anadditional VHH or domain antibody, respectively (in such bindingmolecules, the two or more immunoglobulin single variable domains may belinked directly to said additional immunoglobulin single variable domainor via suitable linkers). Such an additional VHH or domain antibody mayfor example be a VHH or domain antibody that provides for an increasedhalf-life. For example, the latter VHH or domain antibody may be onethat is capable of binding to a (human) serum protein such as (human)serum albumin or (human) transferrin.

Alternatively, the two or more immunoglobulin single variable domainsthat bind to the respective target may be linked in series (eitherdirectly or via a suitable linker) and the additional VHH or domainantibody (which may provide for increased half-life) may be connecteddirectly or via a linker to one of these two or more aforementionedimmunoglobulin sequences.

Suitable linkers are described herein in connection with specificpolypeptides of the invention and may—for example and withoutlimitation—comprise an amino acid sequence, which amino acid sequencepreferably has a length of 9 or more amino acids, more preferably atleast 17 amino acids, such as about 20 to 40 amino acids. However, theupper limit is not critical but is chosen for reasons of convenienceregarding e.g. biopharmaceutical production of such polypeptides.

The linker sequence may be a naturally occurring sequence or anon-naturally occurring sequence. If used for therapeutic purposes, thelinker is preferably non-immunogenic in the subject to which thebispecific binding molecule of the invention is administered.

One useful group of linker sequences are linkers derived from the hingeregion of heavy chain antibodies as described in WO1996/34103 andWO1994/04678.

Other examples are poly-alanine linker sequences such as Ala-Ala-Ala.Further preferred examples of linker sequences are Gly/Ser linkers ofdifferent length such as (gly_(x)ser_(y))_(z) linkers, including(gly₄ser)₃, (gly₄ser)₄, (gly₄ser), (gly₃ser), gly₃, and (gly₃ser₂)₃.

Some non-limiting examples of linkers are contained in bispecificbinding molecules of the invention shown in Table 15 (SEQ ID NOs128-168), e.g. the linkers

(35GS; SEQ ID NO: 169) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS;(9GS; SEQ ID NO: 170) GGGGSGGGS; (40GS; SEQ ID NO: 171)GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS.

If a formatted bispecific binding molecule of the invention is modifiedby the attachment of a polymer, for example of a polyethylene glycol PEG(polyethylene glycol) moiety, the linker sequence preferably includes anamino acid residue, such as a cysteine or a lysine, allowing suchmodification, e.g. PEGylation, in the linker region.

Examples of linkers useful for PEGylation are:

(“GS9, C5”, SEQ ID NO: 172) GGGGCGGGS; (“GS25, C5, SEQ ID NO: 173)GGGGCGGGGSGGGGSGGGGSGGGGS (“GS27, C14”, SEQ ID NO: 174)GGGSGGGGSGGGGCGGGGSGGGGSGGG, (“GS35, C15”, SEQ ID NO: 175)GGGGSGGGGSGGGGCGGGGSGGGGSGGGGSGGGGS, and (“GS35, C5”, SEQ ID NO: 176)GGGGCGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS.

Furthermore, the linker may also be a poly(ethylene glycol) moiety, asshown in e.g. WO2004/081026.

In another embodiment, the immunoglobulin single variable domains arelinked to each other via another moiety (optionally via one or twolinkers), such as another polypeptide which, in a preferred butnon-limiting embodiment, may be a further immunoglobulin single variabledomain as described above. Such moiety may either be essentiallyinactive or may have a biological effect such as improving the desiredproperties of the polypeptide or may confer one or more additionaldesired properties to the polypeptide. For example, and withoutlimitation, the moiety may improve the half-life of the protein orpolypeptide, and/or may reduce its immunogenicity or improve any otherdesired property.

According to a preferred embodiment, a bispecific binding molecule ofthe invention includes, especially when intended for use or used as atherapeutic agent, a moiety which extends the half-life of thepolypeptide of the invention in serum or other body fluids of a patient.The term “half-life” is defined as the time it takes for the serumconcentration of the (modified) polypeptide to reduce by 50%, in vivo,for example due to degradation of the polypeptide and/or clearanceand/or sequestration by natural mechanisms.

More specifically, such half-life extending moiety can be covalentlylinked to or fused to an immunoglobulin single variable domain and maybe, without limitation, an Fc portion, an albumin moiety, a fragment ofan albumin moiety, an albumin binding moiety, such as an anti-albuminimmunoglobulin single variable domain, a transferrin binding moiety,such as an anti-transferrin immunoglobulin single variable domain, apolyoxyalkylene molecule, such as a polyethylene glycol molecule, analbumin binding peptide or a hydroxyethyl starch (HES) derivative.

In another embodiment, the bispecific binding molecule of the inventioncomprises a moiety which binds to an antigen found in blood, such asserum albumin, serum immunoglobulins, thyroxine-binding protein,fibrinogen or transferrin, thereby conferring an increased half-life invivo to the resulting polypeptide of the invention. According to aspecifically preferred embodiment, such moiety is an albumin-bindingimmunoglobulin and, especially preferred, an albumin-bindingimmunoglobulin single variable domain such as an albumin-binding VHHdomain.

If intended for use in humans, such albumin-binding immunoglobulinsingle variable domain preferably binds to human serum albumin andpreferably is a humanized albumin-binding VHH domain;

Immunoglobulin single variable domains binding to human serum albuminare known in the art and are described in further detail in e.g.WO2006/122786. Specifically, useful albumin binding VHHs are ALB 1 andits humanized counterpart, ALB 8 (WO2009/095489). Other albumin bindingVHH domains mentioned in the above patent publication may, however, beused as well.

A specifically useful albumin binding VHH domain is ALB8 which consistsof or contains the amino acid sequence shown in SEQ ID NO: 98 or 254.

According to a further embodiment of the invention, the twoimmunoglobulin single variable domains, in preferably VHHs, may be fusedto a serum albumin molecule, such as described e.g. in WO02001/79271 andWO2003/59934. As e.g. described in WO02001/79271, the fusion protein maybe obtained by conventional recombinant technology: a DNA moleculecoding for serum albumin, or a fragment thereof, is joined to the DNAcoding for the bispecific binding molecule, the obtained construct isinserted into a plasmid suitable for expression in the selected hostcell, e.g. a yeast cell like Pichia pastoris or a bacterial cell, andthe host cell is then transfected with the fused nucleotide sequence andgrown under suitable conditions. The sequence of a useful HSA is shownin SEQ ID NO: 99.

According to another embodiment, a half-life extending modification of apolypeptide of the invention (such modification also reducingimmunogenicity of the polypeptide) comprises attachment of a suitablepharmacologically acceptable polymer, such as straight or branched chainpoly(ethylene glycol) (PEG) or derivatives thereof (such asmethoxypoly(ethylene glycol) or mPEG). Generally, any suitable form ofPEGylation can be used, such as the PEGylation used in the art forantibodies and antibody fragments (including but not limited to domainantibodies and scFv's); reference is made, for example, to: Chapman,Nat. Biotechnol., 54, 531-545 (2002); Veronese and Harris, Adv. DrugDeliv. Rev. 54, 453-456 (2003); Harris and Chess, Nat. Rev. Drug.Discov. 2 (2003); and WO2004/060965.

Various reagents for PEGylation of polypeptides are also commerciallyavailable, for example from Nektar Therapeutics, USA, or NOFCorporation, Japan, such as the Sunbright® EA Series, SH Series, MASeries, CA Series, and ME Series, such as Sunbright® ME-100MA,Sunbright® ME-200MA, and Sunbright® ME-400MA.

Preferably, site-directed PEGylation is used, in particular via acysteine-residue (see for example Yang et al., Protein Engineering 16,761-770 (2003)). For example, for this purpose, PEG may be attached to acysteine residue that naturally occurs in a polypeptide of theinvention, a polypeptide of the invention may be modified so as tosuitably introduce one or more cysteine residues for attachment of PEG,or an amino acid sequence comprising one or more cysteine residues forattachment of PEG may be fused to the N- and/or C-terminus of apolypeptide of the invention, all using techniques of proteinengineering known per se to the skilled person.

Preferably, for the polypeptides of the invention, a PEG is used with amolecular weight of more than 5 kDa, such as more than 10 kDa and lessthan 200 kDa, such as less than 100 kDa; for example in the range of 20kDa to 80 kDa.

With regard to PEGylation, its should be noted that generally, theinvention also encompasses any bispecific binding molecule that has beenPEGylated at one or more amino acid positions, preferably in such a waythat said PEGylation either (1) increases the half-life in vivo; (2)reduces immunogenicity; (3) provides one or more further beneficialproperties known per se for PEGylation; (4) does not essentially affectthe affinity of the polypeptide for its target (e.g. does not reducesaid affinity by more than 50%, and more preferably not by more than10%, as determined by a suitable assay described in the art); and/or (4)does not affect any of the other desired properties of the bispecificbinding molecules of the invention. Suitable PEG-groups and methods forattaching them, either specifically or non-specifically, will be clearto the skilled person. Various reagents for PEGylation of polypeptidesare also commercially available, for example from Nektar Therapeutics,USA, or NOF Corporation, Japan, such as the Sunbright® EA Series, SHSeries, MA Series, CA Series, and ME Series, such as Sunbright®ME-100MA, Sunbright® ME-200MA, and Sunbright® ME-400MA.

According to an especially preferred embodiment of the invention, aPEGylated polypeptide of the invention includes one PEG moiety of linearPEG having a molecular weight of 40 kDa or 60 kDa, wherein the PEGmoiety is attached to the polypeptide in a linker region and,specifically, at a Cys residue at position 5 of a GS9-linker peptide asshown in SEQ ID NO:93, at position 14 of a GS27-linker peptide as shownin SEQ ID NO:95, or at position 15 of a GS35-linker peptide as shown inSEQ ID NO:96, or at position 5 of a 35GS-linker peptide as shown in SEQID NO:97.

A bispecific binding molecule of the invention may be PEGylated with oneof the PEG reagents as mentioned above, such as “Sunbright® ME-400MA”,as shown in the following chemical formula:

Bispecific binding molecules that contain linkers and/or half-lifeextending functional groups are shown in SEQ ID NO: 81 and in FIG. 48.

According to another embodiment, the immunoglobulin single variabledomains are domain antibodies, as defined herein.

Immunoglobulin single variable domains present in the bispecific bindingmolecules of the invention may also have sequences that correspond tothe amino acid sequence of a naturally occurring VH domain that has been“camelized”, i.e. by replacing one or more amino acid residues in theamino acid sequence of a naturally occurring variable heavy chain from aconventional 4-chain antibody by one or more amino acid residues thatoccur at the corresponding position(s) in a VHH domain of a heavy chainantibody. This can be performed in a manner known per se, which will beclear to the skilled person, and reference is additionally be made toWO1994/04678. Such camelization may preferentially occur at amino acidpositions which are present at the VH-VL interface and at the so-calledCamelidae Hallmark residues (see for example also WO1994/04678). Adetailed description of such “humanization” and “camelization”techniques and preferred framework region sequences consistent therewithcan additionally be taken from e.g. pp. 46 and pp. 98 of WO2006/040153and pp. 107 of WO2006/122786.

The binding components have specificity for Ang2 or VEGF, respectively,in that they comprise in a preferred embodiment one or moreimmunoglobulin single variable domains specifically binding to one ormore epitopes within the Ang2 molecule or within the VEGF molecule,respectively.

Specific binding of a binding component to its antigen Ang2 or VEGF canbe determined in any suitable manner known per se, including, forexample, the assays described herein, Scatchard analysis and/orcompetitive binding assays, siath as radioimmunoassays (RIA), enzymeimmunoassays (EIA and ELISA) and sandwich competition assays, and thedifferent variants thereof known per se in the art.

With regard to the antigen Ang2 or VEGF, respectively, an immunoglobulinsingle variable domain is not limited with regard to the species. Thus,the immunoglobulin single variable domains preferably bind to human Ang2or to human VEGF, respectively, if intended for therapeutic purposes inhumans. However, immunoglobulin single variable domains that bind toAng2 or VEGF, respectively, from another mammalian species, orpolypeptides containing them, are also within the scope of theinvention. An immunoglobulin single variable domain binding to onespecies form of Ang2 or VEGF may cross-react with the respective antigenfrom one or more other species. For example, immunoglobulin singlevariable domains binding to the human antigen may exhibit crossreactivity with the respective antigen from one or more other species ofprimates and/or with the antigen from one or more species of animalsthat are used in animal models for diseases, for example monkey (inparticular Cynomolgus or Rhesus), mouse, rat, rabbit, pig, dog or) andin particular in animal models for diseases and disorders that can bemodulated by inhibition of Ang2 (such as the species and animal modelsmentioned herein). Immunoglobulin single variable domains of theinvention that show such cross-reactivity are advantageous in a researchand/or drug development, since it allows the immunoglobulin singlevariable domains of the invention to be tested in acknowledged diseasemodels such as monkeys, in particular Cynomolgus or Rhesus, or mice andrats.

Also, the binding components are not limited to or defined by a specificdomain or an antigenic determinant of the antigen against which they aredirected. Preferably, in view of cross-reactivity with one or moreantigen molecules from species other than human that is/are intended foruse as an animal model during development of a therapeutic Ang2NEGFantagonist, a binding component recognizes an epitope in a region of therespective antigen that has a high degree of identity with the humanantigen. By way of example, in view of using a mouse model, an anti-Ang2immunoglobulin single variable domain contained in the bispecificbinding molecules of the invention recognizes an epitope which is,totally or in part, located within the FLD domain of Ang2, which shows ahigh identity between human and mouse.

Preferably, the VEGF-binding component binds to the VEGF isoformsVEGF165 and/or VEGF121.

In another aspect, the invention relates to nucleic acid molecules thatencode bispecific binding molecules of the invention. Such nucleic acidmolecules will also be referred to herein as “nucleic acids of theinvention” and may also be in the form of a genetic construct, asdefined herein. A nucleic acid of the invention may be genomic DNA, cDNAor synthetic DNA (such as DNA with a codon usage that has beenspecifically adapted for expression in the intended host cell or hostorganism). According to one embodiment of the invention, the nucleicacid of the invention is in essentially isolated form, as definedhereabove.

The nucleic acid of the invention may also be in the form of, may bepresent in and/or may be part of a vector, such as for example aplasmid, cosmid or YAC. The vector may especially be an expressionvector, i.e. a vector that can provide for expression of the bispecificbinding molecule in vitro and/or in vivo (i.e. in a suitable host cell,host organism and/or expression system). Such expression vectorgenerally comprises at least one nucleic acid of the invention that isoperably linked to one or more suitable regulatory elements, such aspromoter(s), enhancer(s), terminator(s), and the like. Such elements andtheir selection in view of expression of a specific sequence in aspecific host are common knowledge of the skilled person. Specificexamples of regulatory elements and other elements useful or necessaryfor expressing bispecific binding molecules of the invention, such aspromoters, enhancers, terminators, integration factors, selectionmarkers, leader sequences, reporter genes, and the like, are disclosede.g. on pp. 131 to 133 of WO2006/040153.

The nucleic acids of the invention may be prepared or obtained in amanner known per se (e.g. by automated DNA synthesis and/or recombinantDNA technology), based on the information on the amino acid sequencesfor the polypeptides of the invention given herein, and/or can beisolated from a suitable natural source.

In another aspect, the invention relates to host cells that express orthat are capable of expressing one or more bispecific binding moleculesof the invention; and/or that contain a nucleic acid of the invention.According to a particularly preferred embodiment, said host cells arebacterial cells; other useful cells are yeast cells, fungal cells ormammalian cells.

Suitable bacterial cells include cells from gram-negative bacterialstrains such as strains of Escherichia coli, Proteus, and Pseudomonas,and gram-positive bacterial strains such as strains of Bacillus,Streptomyces, Staphylococcus, and Lactococcus. Suitable fungal cellinclude cells from species of Trichoderma, Neurospora, and Aspergillus.Suitable yeast cells include cells from species of Saccharomyces (forexample Saccharomyces cerevisiae), Schizosaccharomyces (for exampleSchizosaccharomyces pombe), Pichia (for example Pichia pastoris andPichia methanolica), and Hansenula.

Suitable mammalian cells include for example CHO cells, BHK cells, HeLacells, COS cells, and the like. However, amphibian cells, insect cells,plant cells, and any other cells used in the art for the expression ofheterologous proteins can be used as well.

The invention further provides methods of manufacturing a bispecificbinding molecule of the invention, such methods generally comprising thesteps of:

-   -   culturing host cells comprising a nucleic acid capable of        encoding a bispecific binding molecule under conditions that        allow expression of the bispecific binding molecule of the        invention; and    -   recovering or isolating the polypeptide expressed by the host        cells from the culture; and    -   optionally further purifying and/or modifying and/or formulating        the bispecific binding molecule of the invention.

For production on an industrial scale, preferred host organisms includestrains of E. coli, Pichia pastoris, and S. cerevisiae that are suitablefor large scale expression, production and fermentation, and inparticular for large scale pharmaceutical expression, production andfermentation.

The choice of the specific expression system depends in part on therequirement for certain post-translational modifications, morespecifically glycosylation. The production of a bispecific bindingmolecule of the invention for which glycosylation is desired or requiredwould necessitate the use of mammalian expression hosts that have theability to glycosylate the expressed protein. In this respect, it willbe clear to the skilled person that the glycosylation pattern obtained(i.e. the kind, number and position of residues attached) will depend onthe cell or cell line that is used for the expression.

Bispecific binding molecules of the invention may be produced either ina cell as set out above intracellullarly (e.g. in the cytosol, in theperiplasma or in inclusion bodies) and then isolated from the host cellsand optionally further purified; or they can be produced extracellularly(e.g. in the medium in which the host cells are cultured) and thenisolated from the culture medium and optionally further purified.

Methods and reagents used for the recombinant production ofpolypeptides, such as specific suitable expression vectors,transformation or transfection methods, selection markers, methods ofinduction of protein expression, culture conditions, and the like, areknown in the art. Similarly, protein isolation and purificationtechniques useful in a method of manufacture of a polypeptide of theinvention are well known to the skilled person.

In a further aspect, the invention relates to a peptide having an aminoacid sequence of a CDR3 contained in an anti-VEGF-VHH having an aminoacid sequence selected from sequences shown in SEQ ID NOs: 9 to 57 orSEQ ID NOs: 58-127, respectively, and a nucleic acid molecule encodingsame.

These peptides correspond to CDR3s derived from the VHHs of theinvention. They, in particular the nucleic acid molecules encoding them,are useful for CDR grafting in order to replace a CDR3 in animmunoglobulin chain, or for insertion into a non-immunoglobulinscaffold, e.g. a protease inhibitor, DNA-binding protein, cytochromeb562, a helix-bundle protein, a disulfide-bridged peptide, a lipocalinor an anticalin, thus conferring target-binding properties to suchscaffold. The method of CDR-grafting is well known in the art and hasbeen widely used, e.g. for humanizing antibodies (which usuallycomprises grafting the CDRs from a rodent antibody onto the Fvframeworks of a human antibody).

In order to obtain an immunoglobulin or a non-immunoglobulin scaffoldcontaining a CDR3 of the invention, the DNA encoding such molecule maybe obtained according to standard methods of molecular biology, e.g. bygene synthesis, by oligonucleotide annealing or by means of overlappingPCR fragments, as e.g. described by Daugherty et al., 1991, NucleicAcids Research, Vol. 19, 9, 2471-2476. A method for inserting a VHH CDR3into a non-immunoglobulin scaffold has been described by Nicaise et al.,2004, Protein Science, 13, 1882-1891.

The invention further relates to a product or composition containing orcomprising at least one bispecific binding molecule of the invention andoptionally one or more further components of such compositions known perse, i.e. depending on the intended use of the composition.

For pharmaceutical use, a bispecific binding molecule of the inventionmay be formulated as a pharmaceutical preparation or compositioncomprising at least one bispecific binding molecule of the invention andat least one pharmaceutically acceptable carrier, diluent or excipientand/or adjuvant, and optionally one or more further pharmaceuticallyactive polypeptides and/or compounds. By means of non-limiting examples,such a formulation may be in a form suitable for oral administration,for parenteral administration (such as by intravenous, intramuscular orsubcutaneous injection or intravenous infusion), for topicaladministration, for administration by inhalation, by a skin patch, by animplant, by a suppository, etc. Such suitable administration forms—whichmay be solid, semi-solid or liquid, depending on the manner ofadministration—as well as methods and carriers for use in thepreparation thereof, will be clear to the skilled person, and arefurther described herein.

Thus, in a further aspect, the invention relates to a pharmaceuticalcomposition that contains at least one bispecific binding molecule, inparticular one immunoglobulin single variable domain, of the inventionand at least one suitable carrier, diluent or excipient (i.e. suitablefor pharmaceutical use), and optionally one or more further activesubstances.

The bispecific binding molecules of the invention may be formulated andadministered in any suitable manner known per se: Reference, inparticular for the immunoglobulin single variable domains, is forexample made to WO2004/041862, WO2004/041863, WO2004/041865,WO2004/041867 and WO2008/020079, as well as to the standard handbooks,such as Remington's Pharmaceutical Sciences, 18^(th) Ed., MackPublishing Company, USA (1990), Remington, the Science and Practice ofPharmacy, 21^(th) Edition, Lippincott Williams and Wilkins (2005); orthe Handbook of Therapeutic Antibodies (S. Dubel, Ed.), Wiley, Weinheim,2007 (see for example pages 252-255).

For example, an immunoglobulin single variable domain of the inventionmay be formulated and administered in any manner known per se forconventional antibodies and antibody fragments (including ScFv's anddiabodies) and other pharmaceutically active proteins. Such formulationsand methods for preparing the same will be clear to the skilled person,and for example include preparations suitable for parenteraladministration (for example intravenous, intraperitoneal, subcutaneous,intramuscular, intraluminal, intra-arterial or intrathecaladministration) or for topical (i.e. transdermal or intradermal)administration.

Preparations for parenteral administration may for example be sterilesolutions, suspensions, dispersions or emulsions that are suitable forinfusion or injection. Suitable carriers or diluents for suchpreparations for example include, without limitation, sterile water andpharmaceutically acceptable aqueous buffers and solutions such asphysiological phosphate-buffered saline, Ringer's solutions, dextrosesolution, and Hank's solution; water oils; glycerol; ethanol; glycolssuch as propylene glycol or as well as mineral oils, animal oils andvegetable oils, for example peanut oil, soybean oil, as well as suitablemixtures thereof. Usually, aqueous solutions or suspensions will bepreferred.

Thus, the bispecific binding molecule of the invention may besystemically administered, e.g., orally, in combination with apharmaceutically acceptable vehicle such as an inert diluent or anassimilable edible carrier. For oral therapeutic administration, thebispecific binding molecule of the invention may be combined with one ormore excipients and used in the form of ingestible tablets, buccaltablets, troches, capsules, elixirs, suspensions, syrups, wafers, andthe like. Such compositions and preparations should contain at least0.1% of the binding molecule of the invention. Their percentage in thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of the bispecific binding molecule of theinvention in such therapeutically useful compositions is such that aneffective dosage level will be obtained.

The tablets, pills, capsules, and the like may also contain binders,excipients, disintegrating agents, lubricants and sweetening orflavouring agents, for example those mentioned on pages 143-144 ofWO2008/020079. When the unit dosage form is a capsule, it may contain,in addition to materials of the above type, a liquid carrier, such as avegetable oil or a polyethylene glycol. Various other materials may bepresent as coatings or to otherwise modify the physical form of thesolid unit dosage form. For instance, tablets, pills, or capsules may becoated with gelatin, wax, shellac or sugar and the like. A syrup orelixir may contain the binding molecules of the invention, sucrose orfructose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any unit dosage form should bepharmaceutically acceptable and substantially non-toxic in the amountsemployed. In addition, the bispecific binding molecules of the inventionmay be incorporated into sustained-release preparations and devices.

Preparations and formulations for oral administration may also beprovided with an enteric coating that will allow the constructs of theinvention to resist the gastric environment and pass into theintestines. More generally, preparations and formulations for oraladministration may be suitably formulated for delivery into any desiredpart of the gastrointestinal tract. In addition, suitable suppositoriesmay be used for delivery into the gastrointestinal tract.

The bispecific binding molecules of the invention may also beadministered intravenously or intraperitoneally by infusion orinjection, as further described on pages 144 and 145 of WO2008/020079.

For topical administration of the bispecific binding molecules of theinvention, it will generally be desirable to administer them to the skinas compositions or formulations, in combination with a dermatologicallyacceptable carrier, which may be a solid or a liquid, as furtherdescribed on page 145 of WO2008/020079.

Generally, the concentration of the bispecific binding molecules of theinvention in a liquid composition, such as a lotion, will be from about0.1-25 wt-%, preferably from about 0.5-10 wt-%. The concentration in asemi-solid or solid composition such as a gel or a powder will be about0.1-5 wt-%, preferably about 0.5-2.5 wt-%.

The amount of the bispecific binding molecules of the invention requiredfor use in treatment will vary not only with the particular bindingmolecule selected, but also with the route of administration, the natureof the condition being treated and the age and condition of the patientand will be ultimately at the discretion of the attendant physician orclinician. Also, the dosage of the binding molecules of the inventionvaries depending on the target cell, tumor, tissue, graft, or organ.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

An administration regimen may include long-term, daily treatment. By“long-term” is meant at least two weeks and preferably, several weeks,months, or years of duration.

Necessary modifications in this dosage range may be determined by one ofordinary skill in the art using only routine experimentation given theteachings herein. See Remington's Pharmaceutical Sciences (Martin, E.W., ed. 4), Mack Publishing Co., Easton, Pa. The dosage can also beadjusted by the individual physician in the event of any complication.

According to a further embodiment, the invention relates to the use ofbispecific binding molecules, e.g. immunoglobulin single variabledomains, for therapeutic purposes, such as

-   -   for the prevention, treatment and/or alleviation of a disorder,        disease or condition, especially in a human being, that is        associated with VEGF- and/or Ang2-mediated effects on        angiogenesis or that can be prevented, treated or alleviated by        modulating the Notch signaling pathway and/or the Tie2        signalling pathway with a bispecific binding molecule according        to the invention,    -   in a method of treatment of a patient in need of such therapy,        such method comprising administering, to a subject in need        thereof, a pharmaceutically active amount of at least one        bispecific binding molecule of the invention, e.g. an        immunoglobulin single variable domain, or a pharmaceutical        composition containing same;    -   for the preparation of a medicament for the prevention,        treatment or alleviation of disorders, diseases or conditions        associated with VEGF- and/or Ang2-mediated effects on        angiogenesis;    -   as an active ingredient in a pharmaceutical composition or        medicament used for the above purposes.

According to a specific aspect, said disorder, disease or condition is acancer or cancerous disease, as defined herein.

According to another aspect, the disease is an eye disease associatedwith VEGF- and/or Ang2-mediated effects on angiogenesis or which can betreated or alleviated by modulating the Notch signaling pathway with abispecific binding molecule.

Depending on the cancerous disease to be treated, a bispecific bindingmolecule of the invention may be used on its own or in combination withone or more additional therapeutic agents, in particular selected fromchemotherapeutic agents like DNA damaging agents or therapeuticallyactive compounds that inhibit angiogenesis, signal transduction pathwaysor mitotic checkpoints in cancer cells.

The additional therapeutic agent may be administered simultaneouslywith, optionally as a component of the same pharmaceutical preparation,or before or after administration of the binding molecule.

In certain embodiments, the additional therapeutic agent may be, withoutlimitation (and in the case of the receptors, including the respectiveligands), one or more inhibitors selected from the group of inhibitorsof EGFR, VEGFR, HER2-neu, Her3, AuroraA, AuroraB, PLK and PI3 kinase,FGFR, PDGFR, Raf, Ras, KSP, PDK1, PTK2, IGF-R or IR.

Further examples of additional therapeutic agents are inhibitors of CDK,Akt, src/bcr abl, cKit, cMet/HGF, c-Myc, Flt3, HSP90, hedgehogantagonists, inhibitors of JAK/STAT, MEK, mTor, NFkappaB, theproteasome, Rho, an inhibitor of wnt signaling or an inhibitor of theubiquitination pathway or another inhibitor of the Notch signalingpathway.

Examples for Aurora inhibitors are, without limitation, PHA-739358,AZD-1152, AT 9283, CYC-116, R-763, VX-680, VX-667, MLN-8045, PF-3814735.

An example for a PLK inhibitor is GSK-461364.

Examples for raf inhibitors are BAY-73-4506 (also a VEGFR inhibitor),PLX 4032, RAF-265 (also in addition a VEGFR inhibitor), sorafenib (alsoin addition a VEGFR inhibitor), and XL 281.

Examples for KSP inhibitors are ispinesib, ARRY-520, AZD-4877,CK-1122697, GSK 246053A, GSK-923295, MK-0731, and SB-743921.

Examples for a src and/or bcr-abl inhibitors are dasatinib, AZD-0530,bosutinib, XL 228 (also an IGF-1R inhibitor), nilotinib (also a PDGFRand cKit inhibitor), imatinib (also a cKit inhibitor), and NS-187.

An example for a PDK1 inhibitor is BX-517.

An example for a Rho inhibitor is BA-210.

Examples for PI3 kinase inhibitors are PX-866, BEZ-235 (also an mTorinhibitor), XL 418 (also an Akt inhibitor), XL-147, and XL 765 (also anmTor inhibitor).

Examples for inhibitors of cMet or HGF are XL-184 (also an inhibitor ofVEGFR, cKit, Flt3), PF-2341066, MK-2461, XL-880 (also an inhibitor ofVEGFR), MGCD-265 (also an inhibitor of VEGFR, Ron, Tie2), SU-11274,PHA-665752, AMG-102, and AV-299.

An example for a c-Myc inhibitor is CX-3543.

Examples for Flt3 inhibitors are AC-220 (also an inhibitor of cKit andPDGFR), KW 2449, lestaurtinib (also an inhibitor of VEGFR, PDGFR, PKC),TG-101348 (also an inhibitor of JAK2), XL-999 (also an inhibitor ofcKit, FGFR, PDGFR and VEGFR), sunitinib (also an inhibitor of PDGFR,VEGFR and cKit), and tandutinib (also an inhibitor of PDGFR, and cKit).

Examples for HSP90 inhibitors are tanespimycin, alvespimycin, IPI-504and CNF 2024.

Examples for JAK/STAT inhibitors are CYT-997 (also interacting withtubulin), TG 101348 (also an inhibitor of Fit3), and XL-019.

Examples for MEK inhibitors are ARRY-142886, PD-325901, AZD-8330, and XL518.

Examples for mTor inhibitors are temsirolimus, AP-23573 (which also actsas a VEGF inhibitor), everolimus (a VEGF inhibitor in addition). XL-765(also a PI3 kinase inhibitor), and BEZ-235 (also a PI3 kinaseinhibitor).

Examples for Akt inhibitors are perifosine, GSK-690693, RX-0201, andtriciribine.

Examples for cKit inhibitors are AB-1010, OSI-930 (also acts as a VEGFRinhibitor), AC-220 (also an inhibitor of Flt3 and PDGFR), tandutinib(also an inhibitor of Flt3 and PDGFR), axitinib (also an inhibitor ofVEGFR and PDGFR), XL-999 (also an inhibitor of Flt3, PDGFR, VEGFR,FGFR), sunitinib (also an inhibitor of Flt3, PDGFR, VEGFR), and XL-820(also acts as a VEGFR- and PDGFR inhibitor), imatinib (also a bcr-ablinhibitor), nilotinib (also an inhibitor of bcr-abl and PDGFR).

Examples for hedgehog antagonists are IPI-609 and CUR-61414.

Examples for CDK inhibitors are seliciclib, AT-7519, P-276, ZK-CDK (alsoinhibiting VEGFR2 and PDGFR), PD-332991, R-547, SNS-032, PHA-690509, andAG 024322.

Examples for proteasome inhibitors are bortezomib, carfilzomib, andNPI-0052 (also an inhibitor of NFkappaB).

An example for an NFkappaB pathway inhibitor is NPI-0052.

An example for an ubiquitination pathway inhibitor is HBX-41108.

In preferred embodiments, the additional therapeutic agent is ananti-angiogenic agent.

Examples for anti-angiogenic agents are inhibitors of the FGFR, PDGFRand VEGFR or the respective ligands (e.g VEGF inhibitors like pegaptanibor the anti-VEGF antibody bevacizumab), EGFL7 inhibitors, such asanti-EGFL7 MAb, angiopoietinl/2 inhibitors such as AMG386, andthalidomides, such agents being selected from, without limitation,bevacizumab, motesanib, CDP-791, SU-14813, telatinib, KRN-951, ZK-CDK(also an inhibitor of CDK), ABT-869, BMS-690514, RAF-265, IMC-KDR,IMC-18F1, IMiDs (immunomodulatory drugs), thalidomide derivativeCC-4047, lenalidomide, ENMD 0995, IMC-D11, Ki 23057, brivanib,cediranib, XL-999 (also an inhibitor of cKit and Flt3), 1B3, CP 868596,IMC 3G3, R-1530 (also an inhibitor of Flt3), sunitinib (also aninhibitor of cKit and Flt3), axitinib (also an inhibitor of cKit),lestaurtinib (also an inhibitor of Flt3 and PKC), vatalanib, tandutinib(also an inhibitor of Fit3 and cKit), pazopanib, GW 786034, PF-337210,IMC-1121B, AVE-0005, AG-13736, E-7080, CHIR 258, sorafenib tosylate(also an inhibitor of Raf), RAF-265 (also an inhibitor of Raf),vandetanib, CP-547632, OSI-930, AEE-788 (also an inhibitor of EGFR andHer2), BAY-57-9352 (also an inhibitor of Raf), BAY-73-4506 (also aninhibitor of Raf), XL 880 (also an inhibitor of cMet), XL-647 (also aninhibitor of EGFR and EphB4), XL 820 (also an inhibitor of cKit), andnilotinib (also an inhibitor of cKit and brc-abl).

The additional therapeutic agent may also be selected from EGFRinhibitors, it may be a small molecule EGFR inhibitor or an anti-EGFRantibody. Examples for anti-EGFR antibodies, without limitation, arecetuximab, panitumumab, matuzumab; an example for a small molecule EGFRinhibitor is gefitinib. Another example for an EGFR modulator is the EGFfusion toxin.

Among the EGFR and Her2 inhibitors useful for combination with thebispecific binding molecule of the invention are lapatinib, gefitinib,erlotinib, cetuximab, trastuzumab, nimotuzumab, zalutumumab, vandetanib(also an inhibitor of VEGFR), pertuzumab, XL-647, HKI-272, BMS-599626ARRY-334543, AV 412, mAB-806, BMS-690514, JNJ-26483327, AEE-788 (also aninhibitor of VEGFR), ARRY-333786, IMC-11F8, Zemab.

Other agents that may be advantageously combined in a therapy with thebispecific binding molecule of the invention are tositumumab andibritumomab tiuxetan (two radiolabelled anti-CD₂₀ antibodies),alemtuzumab (an anti-CD52 antibody), denosumab, (an osteoclastdifferentiation factor ligand inhibitor), galiximab (a CD80 antagonist),ofatumumab (a CD₂₀ inhibitor), zanolimumab (a CD4 antagonist), SGN40 (aCD40 ligand receptor modulator), rituximab (a CD₂₀ inhibitor),mapatumumab (a TRAIL-1 receptor agonist), REGN421 (SAR153192) orOMP-21M18 (DII4 inhibitors).

Other chemotherapeutic drugs that may be used in combination with thebispecific binding molecule of the present invention are selected from,but not limited to hormones, hormonal analogues and antihormonals (e.g.tamoxifen, toremifene, raloxifene, fulvestrant, megestrol acetate,flutamide, nilutamide, bicalutamide, cyproterone acetate, finasteride,buserelin acetate, fludrocortisone, fluoxymesterone,medroxyprogesterone, octreotide, arzoxifene, pasireotide, vapreotide),aromatase inhibitors (e.g. anastrozole, letrozole, liarozole,exemestane, atamestane, formestane), LHRH agonists and antagonists (e.g.goserelin acetate, leuprolide, abarelix, cetrorelix, deslorelin,histrelin, triptorelin), antimetabolites (e.g. antifolates likemethotrexate, pemetrexed, pyrimidine analogues like 5 fluorouracil,capecitabine, decitabine, nelarabine, and gemcitabine, purine andadenosine analogues such as mercaptopurine thioguanine, cladribine andpentostatin, cytarabine, fludarabine); antitumor antibiotics (e.g.anthracyclines like doxorubicin, daunorubicin, epirubicin andidarubicin, mitomycin-C, bleomycin dactinomycin, plicamycin,mitoxantrone, pixantrone, streptozocin); platinum derivatives (e.g.cisplatin, oxaliplatin, carboplatin, lobaplatin, satraplatin);alkylating agents (e.g. estramustine, meclorethamine, melphalan,chlorambucil, busulphan, dacarbazine, cyclophosphamide, ifosfamide,hydroxyurea, temozolomide, nitrosoureas such as carmustine andlomustine, thiotepa); antimitotic agents (e.g. vinca alkaloids likevinblastine, vindesine, vinorelbine, vinflunine and vincristine; andtaxanes like paclitaxel, docetaxel and their formulations, larotaxel;simotaxel, and epothilones like ixabepilone, patupilone, ZK-EPO);topoisomerase inhibitors (e.g. epipodophyllotoxins like etoposide andetopophos, teniposide, amsacrine, topotecan, irinotecan) andmiscellaneous chemotherapeutics such as amifostine, anagrelide,interferone alpha, procarbazine, mitotane, and porfimer, bexarotene,celecoxib.

The efficacy of bispecific binding molecule of the invention orpolypeptides, and of compositions comprising the same, can be testedusing any suitable in vitro assay, cell-based assay, in vivo assayand/or animal model known per se, or any combination thereof, dependingon the specific disease or disorder of interest. Suitable assays andanimal models will be clear to the skilled person, and for exampleinclude the assays described herein and used in the Examples below, e.g.a proliferation assay.

The data obtained in the experiments of the invention confirm thatbispecific binding molecules of the invention have properties that aresuperior to those of binding molecules of the prior art. Among suchproperties are complete inhibition of the VEGF165-VEGFR2 interaction anda low IC50, as can e.g. be taken from the ELISA data of FIG. 1 and Table5 as well as the IC₅₀ (nM) values for VHHs in the AlphaScreen assay asshown in FIGS. 3, 17, 18 and Table 7; and the affinity K_(D) (nM) ofpurified VHHs on recombinant human VEGF and mouse VEGF in Table 9, 10and FIG. 5. Also, as shown in Table 13, VEGF binders of the inventionhave high potency, i.e. in the subnanomolar range, in the HUVECproliferation assay. This indicates that bispecific binding molecules ofthe invention are promising candidates to have therapeutic efficacy indiseases and disorders associated with VEGF-mediated effects onangiogenesis, such as cancer.

According to another embodiment of the invention, there is provided amethod of diagnosing a disease by

-   a) contacting a sample with a binding molecule of the invention as    defined above, and-   b) detecting binding of said binding molecule to said sample, and-   c) comparing the binding detected in step (b) with a standard,    wherein a difference in binding relative to said sample is    diagnostic of a disease or disorder associated with VEGF- and/or    Ang2-mediated effects on angiogenesis.

For this and other uses, it may be useful to further modify a bispecificbinding molecule of the invention, such as by introduction of afunctional group that is one part of a specific binding pair, such asthe biotin-(strept)avidin binding pair. Such a functional group may beused to link the binding molecule of the invention to another protein,polypeptide or chemical compound that is bound to the other half of thebinding pair, i.e. through formation of the binding pair. For example, abispecific binding molecule of the invention may be conjugated tobiotin, and linked to another protein, polypeptide, compound or carrierconjugated to avidin or streptavidin. For example, such a conjugatedbispecific binding molecule of the invention may be used as a reporter,for example in a diagnostic system where a detectable signal-producingagent is conjugated to avidin or streptavidin.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Purified monovalent VHHs block the hVEGF165/hVEGFR2-Fcinteraction (ELISA)

FIG. 2: Purified monovalent VHHs block the hVEGF165/hVEGFR1-Fcinteraction (ELISA)

FIG. 3: Purified monovalent VHHs block the hVEGF165/hVEGFR2-Fcinteraction (AlphaScreen)

FIG. 4: Purified monovalent VHHs block the hVEGF165/hVEGFR1-Fcinteraction (AlphaScreen)

FIG. 5: Binding of monovalent VHHs to recombinant human and mouse VEGF(ELISA)

FIG. 6: Binding of monovalent VHHs to human VEGF121

FIG. 7: Purified VHHs do not bind to VEGFB, VEGFC, VEGFD and PIGF

FIG. 8: Formatted VHHs block hVEGF165/hVEGFR2-Fc interaction (ELISA)

FIG. 9: Formatted VHHs block hVEGF165/hVEGFR1-Fc interaction (ELISA)

FIG. 10: Formatted VHHs block hVEGF165/hVEGFR2-Fc interaction(AlphaScreen)

FIG. 11: Formatted VHHs block hVEGF165/hVEGFR1-Fc interaction(AlphaScreen)

FIG. 12: Formatted VHHs block mVEGF164/mVEGFR2-Fc interaction(AlphaScreen)

FIG. 13: Formatted VHHs bind to mouse and human VEGF

FIG. 14: Formatted VHHs do not bind to VEGFB, VEGFC, VEGFD and PIGF

FIG. 15: Formatted VHHs bind to VEGF121

FIG. 16: Sequence alignment of VHH VEGFBII23B04 with human VH3/JHgermline consensus sequence

FIG. 17: VHH variants of VEGFBII23B04 block hVEGF165/hVEGFR2-Fcinteraction (AlphaScreen)

FIG. 18: Sequence-optimized clones of VEGFBII23B04 block thehVEGF165/hVEGFR2-Fc interaction (AlphaScreen)

FIG. 19: Sequence alignment of VHH VEGFBII5B05 with human VH3/JHgermline consensus sequence

FIG. 20: Description bivalent Ang2 VHHs

FIG. 21: Purified bivalent Ang2 VHHs blocking hAng2-hTie2 (25-1),mAng2-mTie2 (25-2) and cAng2-cTie2 (25-3) interaction (ELISA)

FIG. 22: Purified bivalent Ang2 VHHs blocking hAng1-hTie2 interaction(ELISA)

FIG. 23: Description trivalent VEGFxAng2 bispecific VHHs

FIG. 24: Purified trivalent VEGFxAng2 Nanobodies blocking hVEGF-hVEGFR2interaction (AlphaScreen)

FIG. 25: Purified trivalent VEGFxAng2 VHHs blocking hAng2-hTie2interaction (ELISA)

FIG. 26: Description trivalent and tetravalent VEGFxAng2 bispecific VHHs

FIG. 27: Purified trivalent and tetravalent VEGFxAng2 VHHs blockinghVEGF-hVEGFR2 (31-1) and hVEGF-hVEGFR1 (31-2) interaction (AlphaScreen)

FIG. 28: Purified trivalent and tetravalent VEGFxAng2 VHHs blockinghAng2-hTie2 (32-1), mAng2-mTie2 (32-2) and cAng2-cTie2 (32-3)interaction (ELISA)

FIG. 29: Purified trivalent and tetravalent VEGFxAng2 VHHs blockinghAng2 mediated HUVEC survival

FIG. 30: Description sequence optimized and affinity VEGFxAng2bispecific VHHs

FIG. 31: Purified VEGFANGBII00022-25-28 VEGFxAng2 VHHs blockinghVEGF-hVEGFR2 (35-1) and hVEGF-hVEGFR1 (35-2) interaction (AlphaScreen)

FIG. 32: Purified VEGFANGBII00022-25-28 VEGFxAng2 VHHs binding to humanVEGF165 (36-1) and hVEGF121 (36-2) (ELISA)

FIG. 33: Purified VEGFANGBII00022-25-28 VEGFxAng2 VHHs binding to (A)mouse and (B) rat VEGF164 (ELISA)

FIG. 34: Purified VEGFANGBII00022-25-28 VEGFxAng2 VHHs binding to (A)human VEGF-B, (B) human VEGF-C, (C) human VEGF-D and (D) human PIGF(ELISA)

FIG. 35: Purified VEGFANGBII00022-25-28 VEGFxAng2 VHHs blockinghAng2-hTie2 (39-1), mAng2-mTie2 (39-2) and cAng2-cTie2 (39-3)interaction (ELISA)

FIG. 36: Purified VEGFANGBII00022-25-28 VEGFxAng2 VHHs blockinghAng1-hTie2 interaction (ELISA)

FIG. 37: Purified VEGFANGBII00022-25-28 VEGFxAng2 VHHs blocking hAng2mediated HUVEC survival

MATERIALS AND METHODS a) Production and Functionality Testing of VEGF109

A cDNA encoding the receptor binding domain of human vascularendothelial growth factor isoform VEGF165 (GenBank: AAM03108.1; AAresidues 27-0.135) is cloned into pET28a vector (Novagen, Madison, Wis.)and overexpressed in E. coli (BL21 Star DE3) as a His-tagged insolubleprotein. Expression is induced by addition of 1 mM IPTG and allowed tocontinue for 4 hours at 37° C. Cells are harvested by centrifugation andlysed by sonication of the cell pellet. Inclusion bodies are isolated bycentrifugation. After a washing step with 1% Triton X 100(Sigma-Aldrich), proteins are solubilized using 7.5M guanidinehydrochloride and refolded by consecutive rounds of overnight dialysisusing buffers with decreasing urea concentrations from 6M till 0M. Therefolded protein is purified by ion exchange chromatography using aMonoQ5/50 GL (Amersham BioSciences) column followed by gel filtrationwith a Superdex75 10/300 GL column (Amersheim BioSciences). The purityand homogeneity of the protein is confirmed by SDS-PAGE and Westen blot.In addition, binding activity to VEGFR1, VEGFR2 and Bevacizumab ismonitored by ELISA. To this end, 1 μg/mL of recombinant human VEGF109 isimmobilized overnight at 4° C. in a 96-well MaxiSorp plate (Nunc,Wiesbaden, Germany). Wells are blocked with a casein solution (1%).Serial dilutions of VEGFR1, VEGFR2 or Bevacizumab are added to theVEGF109 coated plate and binding is detected using alkaline phosphatase(AP) conjugated goat anti-human IgG, Fc specific (Jackson ImmunoResearch Laboratories Inc., West Grove, Pa., USA) and a subsequentenzymatic reaction in the presence of the substrate PNPP(p-nitrophenylphosphate) (Sigma-Aldrich). VEGF109 could bind to VEGFR1,VEGFR2 and Bevacizumab, indicating that the produced VEGF109 is active.

b) KLH Conjugation of VEGF165 and Functionality Testing ofKLH-Conjugated VEGF165

Recombinant human VEGF165 (R&D Systems, Minneapolis, Minn., USA) isconjugated to mariculture keyhole limpet hemocyanin (mcKLH) using theImject Immunogen EDC kit with mcKLH (Pierce, Rockford, Ill., USA)according to the manufacturer's instructions. Efficient conjugation ofthe polypeptide to mcKLH is confirmed by SDS-PAGE.

Functionality of the conjugated protein is checked by ELISA: 2 μg/mL ofKLH conjugated VEGF165 is immobilized overnight at 4° C. in a 96-wellMaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with acasein solution (1%). Serial dilutions of VEGFR1, or VEGFR2 are addedand binding is detected using a horseradish peroxidase (HRP)-conjugatedgoat anti-human IgG, Fc specific (Jackson Immuno Research LaboratoriesInc., West Grove, Pa., USA) and a subsequent enzymatic reaction in thepresence of the substrate TMB (3,3′,5,5′-tetramentylbenzidine) (Pierce,Rockford, Ill., USA). The KLH conjugated protein could still interactwith VEGFR1, VEGFR2 and Bevacizumab, confirming that the relevantepitopes on VEGF165 are still accessible.

Example 1 Immunization with Different VEGF Formats Induces a HumoralImmune Response in Llama 1.1 Immunizations

After approval of the Ethical Committee of the faculty of VeterinaryMedicine (University Ghent, Belgium), 4 llamas (designated No. 264, 265,266, 267) are immunized according to standard protocols with 6intramuscular injections (100 or 50 μg/dose at weekly intervals) ofrecombinant human VEGF109. The first injection at day 0 is formulated inComplete Freund's Adjuvant (Difco, Detroit, Mich., USA), while thesubsequent injections are formulated in Incomplete Freund's Adjuvant(Difco, Detroit, Mich., USA). In addition, four llamas (designated No.234, 235, 280 and 281) are immunized according to the followingprotocol: 5 intramuscular injections with KLH-conjugated human VEGH165(100 or 50 μg/dose at biweekly intervals) followed by 4 intramuscularinjections of human VEGF109 (first dose of 100 μg followed 2 weeks laterwith three 50 μg/dose at weekly interval).

1.2 Evaluation of VEGF-Induced Immune Responses in Llama

To monitor VEGF specific serum titers, an ELISA assay is set up in which2 μg/mL of recombinant human VEGF165 or VEGF109 is immobilized overnightat 4° C. in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wellsare blocked with a casein solution (1%). After addition of serumdilutions, bound total IgG is detected using horseradish peroxidase(HRP)-conjugated goat anti-llama immunoglobulin (Bethyl LaboratoriesInc., Montgomery, Tex., USA) and a subsequent enzymatic reaction in thepresence of the substrate TMB (3,3′,5,5′-tetramentylbenzidine) (Pierce,Rockford, Ill., USA). For llamas 264, 265, 266 and 267, an additionalELISA is performed in which the isotype-specific responses againstVEGF165 and VEGF109 are evaluated. Isotype specific responses aredetected using mouse mAbs specifically recognizing conventional llamaIgG1 and the heavy-chain only llama IgG2 and IgG3 [Daley et al. (2005).Clin. Diagn. Lab. Imm. 12:380-386] followed by a rabbit anti-mouse-HRPconjugate (DAKO). ELISAs are developed using TMB as chromogenicsubstrate and absorbance is measured at 450 nm. The serum titers foreach llama are depicted in Table 1.

TABLE 1 Antibody-mediated specific serum response against VEGF165 andVEGF109 ELISA (recombinant protein solid phase coated) Recombinant humanEGF165 Recombinant human VEGF109 Llama Immunogen Total IgG IgG1 IgG2IgG3 Total IgG IgG1 IgG2 IgG3 234 VEGF165-KLH + ++ n/d n/d n/d ++ n/dn/d n/d VEGF109 235 VEGF165-KLH + ++ n/d n/d n/d ++ n/d n/d n/d VEGF109280 VEGF165-KLH + + n/d n/d n/d + n/d n/d n/d VEGF109 281VEGF165-KLH + + n/d n/d n/d + n/d n/d n/d VEGF109 264 VEGF109 n/d ++ + +++ ++ + + 265 VEGF109 n/d ++ + + + ++ + + 266 VEGF109 n/d ++ + +/− ++++ + +/− 267 VEGF109 n/d +/− − − +/− +/− − − n/d, not determined

Example 2 Cloning of the Heavy-Chain Only Antibody Fragment Repertoiresand Preparation of Phage

Following the final immunogen injection, immune tissues as the source ofB-cells that produce the heavy-chain antibodies are collected from theimmunized llamas. Typically, two 150-ml blood samples, collected 4 and 8days after the last antigen injection, and one lymph node biopsy,collected 4 days after the last antigen injection are collected peranimal. From the blood samples, peripheral blood mononuclear cells(PBMCs) are prepared using Ficoll-Hypaque according to themanufacturer's instructions (Amersham Biosciences, Piscataway, N.J.,USA). From the PBMCs and the lymph node biopsy, total RNA is extracted,which is used as starting material for RT-PCR to amplify the VHHencoding DNA segments, as described in WO2005/044858. For each immunizedllama, a library is constructed by pooling the total RNA isolated fromall collected immune tissues of that animal. In short, the PCR-amplifiedVHH repertoire is cloned via specific restriction sites into a vectordesigned to facilitate phage display of the VHH library. The vector isderived from pUC119 and contains the LacZ promoter, a M13 phage gillprotein coding sequence, a resistance gene for ampicillin orcarbenicillin, a multiple cloning site and a hybrid gill-pelB leadersequence (pAX050). In frame with the VHH coding sequence, the vectorencodes a C-terminal c-myc tag and a His6 tag. Phage are preparedaccording to standard protocols and stored after filter sterilization at4° C. for further use.

Example 3 Selection of VEGF-Specific VHHs Via Phage Display

VHH phage libraries are used in different selection strategies applyinga multiplicity of selection conditions. Variables include i) the VEGFprotein format (rhVEGF165, rhVEGF109 or rmVEGF164), ii) the antigenpresentation method (solid phase: directly coated or via a biotin-tagonto Neutravidin-coated plates; solution phase: incubation in solutionfollowed by capturing on Neutravidin-coated plates), iii) the antigenconcentration and iv) the elution method (trypsin or competitive elutionusing VEGFR2). All selections are carried out in Maxisorp 96-well plates(Nunc, Wiesbaden, Germany).

Selections are performed as follows: Phage libraries are incubated at RTwith variable concentrations of VEGF antigen, either in solution orimmobilized on a solid support. After 2 hrs of incubation and extensivewashing, bound phage are eluted. In case trypsin is used for phageelution, the protease activity is immediately neutralized by addition of0.8 mM protease inhibitor AEBSF. Phage outputs that show enrichment overbackground are used to infect E. coli. Infected E. coli cells are eitherused to prepare phage for the next selection round (phage rescue) orplated on agar plates (LB+amp+glucose^(2%)) for analysis of individualVHH clones. In order to screen a selection output for specific binders,single colonies are picked from the agar plates and grown in 1 mL96-deep-well plates. The lacZ-controlled VHH expression is induced byadding IPTG (0.1-1 mM final). Periplasmic extracts (in a volume of −80μL) are prepared according to standard methods.

Example 4 Identification of VEGF-Binding and VEGF Receptor-Blocking VHHs

Periplasmic extracts are tested for binding to human VEGF165 by ELISA.In brief, 2 μg/mL of recombinant human VEGF165 is immobilized overnightat 4° C. in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wellsare blocked with a casein solution (1%). After addition of typically a10-fold dilution of the periplasmic extracts, VHH binding is detectedusing a mouse anti-myc (Roche) and an anti-mouse-HRP conjugate (DAKO).Clones showing ELISA signals of ≧3-fold above background are consideredas VEGF binding VHHs.

In addition, periplasmic extracts are screened in a human VEGF165/humanVEGFR2 AlphaScreen assay (Amplified Luminescent Proximity HomogeneousAssay) to assess the blocking capacity of the VHHs. Human VEGF165 isbiotinylated using Sulfo-NHS-LC-Biotin (Pierce, Rockford, Ill., USA).Human VEGFR2/Fc chimera (R&D Systems, Minneapolis, Minn., USA) iscaptured using an anti-humanFc VHH which is coupled to acceptor beadsaccording to the manufacturer's instructions (Perkin Elmer, Waltham,Mass., US). To evaluate the neutralizing capacity of the VHHs,periplasmic extracts are to diluted 1/25 in PBS buffer containing 0.03%Tween 20 (Sigma-Aldrich) and preincubated with 0.4 nM biotinylated humanVEGF165 for 15 minutes at room temperature (RT). To this mixture theacceptor beads (10 μg/ml) and 0.4 nM VEGFR2-huFc are added and furtherincubated for 1 hour at RT in the dark. Subsequently donor beads (10μg/ml) are added followed by incubation of 1 hour at RT in the dark.Fluorescence is measured by reading plates on the Envision Multi labelPlate reader (Perkin Elmer, Waltham, Mass., USA) using an excitationwavelength of 680 nm and an emission wavelength between 520 nm and 620nm. Periplasmic extract containing irrelevant VHH is used as negativecontrol. Periplasmic extracts containing anti-VEGF165 VHHs which areable to decrease the fluorescence signal with more than 60% relative tothe signal of the negative control are identified as a hit. All hitsidentified in the AlphaScreen are confirmed in a competition ELISA. Tothis end, 1 μg/mL of human VEGFR2 chimera (R&D Systems, Minneapolis,Minn., USA) is coated in a 96-well MaxiSorp plate (Nunc, Wiesbaden,Germany). Fivefold dilutions of the periplasmic extracts are incubatedin the presence of a fixed concentration (4 nM) of biotinylated humanVEGF165 in PBS buffer containing 0.1% casein and 0.05% Tween 20(Sigma-Aldrich). Binding of these VHH/bio-VEGF165 complexes to the humanVEGFR2 chimera coated plate is detected using horseradish peroxidase(HRP) conjugated extravidin (Sigma, St Louis, Mo., USA). VHH sequenceIDs and the corresponding AA sequences of VEGF-binding(non-receptor-blocking) VHHs and inhibitory (receptor-blocking) VHHs arelisted in Table 2 and Table 3, respectively.

TABLE 2 Sequence IDs and AA sequences of monovalent “non-receptor-blocking”anti-VEGF VHHs (FR, framework; CDR, complementary determining region)VHH ID/ SEQ ID NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 VEGFBII01C02/EVQLVESGGG SYGMG WFRQSPG AISEYSNTY RFTISRDNTKNTV SPTILLTTEQWYK WGQGTQ 58LVQAGGSLRL KEREFVS CSDSVRG YLQMNSLTPDDTA Y VTVSS SCTASGGSFS IYYCAAVEGFBII01E07/ EVQLVESGGG ASDMG WFRQAPG AINWSGLST RFTISRDNDNGALGRIPSSSRFSSPA WGQGTQ 59 LVQAGDSLRL KEREFVA FYTDSVKG YLQMNTLKPEDTA AYASVTVSS SCVATGRTFR VYSCAA VEGFBII03D12/ EVQLVESGGG ITVMA WFRQAPG AITWSAPTTRFTISRDNAKNTV DRFKGRSIVTPSD WGQGTQ 60 LVQAGGSLRL KEREFVA YYADSVKGYLRMNSLKPEDSA YRY VTVSS SCTASTSIYT IYYCAA VEGFBII04B08/ EVQLVESGGG DITVAWYRQAPG TITPSGYTY RFTISRDNSKNIV QFY WGQGTQ 61 LVQPGGSLRL IQRQLVA YWDFVKGYLQMNSLKPEDTA VTVSS SCAASGSAVG AYYCNT VEGFBII05B02/ EVQLVESGGG TDDVGWFRQAPG VIRWSTGGT RFTLSRDNAKNTM RSRPLGAGAWYSG WGQGTQ 62 LVQAGGSLRLKEREFVA YTSDSVKG YLQMNSLKPEDTA EKHYNY VTVSS SCAASGRTFS VYYCAAVEGFBII05B03/ EVQLVESGGG HYNMG WFRQAPG SIRGGGGST RFTISRENAKNTVTAFYRGPYDYDY WGQGTQ 63 LAQAGDSLRL KEREFVA TYANSVKD YLQMNSLKPEDTA VTVSSSCAASGRSFS VYYCAA VEGFBII05B05/ EVQLVESGGG SMA WYRQAPG RISSGGTTARFTISRDNSKNTV FSSRPNP WGAGTQ 64 LVQPGGSLRL KHRELVA YVDSVKG YLQMNSLKPEDTAVTVSS SCVASGIRFM VYYCNT VEGFBIIL06G02/ EVQLVESGGG NNAMA WYRQAPGRISSGGGFT RFTVSRDNAKNTV AYRTYNY WGQGTQ 65 LVQPGGSLRL KQRELVA YYLDSVKGYLQMNSLKPEDTA VTVSS SCAASGNIFS VYYCNA VEGFBII07A03/ EVQLVESGGG ITVMAWFRQAPG AITWSAPSS RFTISRDNAKNTV DRFKGRSIVTRSD WGQGTQ 66 LVQAGGSLRLKESEFVA YYADSVKG YLQMNSLKPEDSA YKY VTVSS SCAASTSIYS IYYCAA VEGFBII07A06/EVQLVESGGG ISVMA WFRQAPG AITWSAPTT RFTISRDNAKNTV DRFKGRSIVTRSD WGQGTQ 67LVQAGGSLRL KERAFVA YYADSVKG YLQTNSLKPEDSA YRY VTVSS SCAVSTSIYS IYYCAAVEGFBII07D08/ EVQLVESGGG NYAMA WFRQAPG AINQRGSNT RFTISRDSAKNSVSTWYGYSTYARRE WGQGTQ 68 LVQTGGSLRL KEREFVS NYADSVKG FLQMNSLKPEDTA EYRYVTVSS SCAASGRTFS VYYCAA VEGFBII08D09/ EVQLVESGGG DNVMG WFRQAAG HISRGGSRTRFTISRDNTKKTM SRSVALATARPYD WGQGTQ 69 LVQAGGSLRL KEREFVA EYAESVKGYLQMNSLKPEDTA Y VTVSS SCAASGRSFS VYYCAA VEGFBII08E07/ EVQLVESGGG SYYMGWFRQAPG TISWNKIST RFTVSRDNNKNTV DASRPTLRIPQY WGQGTQ 70 LAQAGGSLRLKEREFVA IYTDSVKG YLQMNSLKPEDTA VTVSS SCTTSGLTFS VYYCAA VEGFBII08F06/EVQLVESGGG SDVMG WYRQAPG FIRSLGSTY RFTISRDDAANTV RFSGESY WGQGTP 71LVQPGGSLRL KQRELVA YAGSVKG YLQMNNLKPEDTA VTVSS SCAASGSIVR  VYYCNA VEGFBII08F07/ EVQLVESGGG LYAMG WFRQAPG AITWSAGDT RFTISRDNARNTVRQWGGTYYYHGSY WGQGTQ 72 LVQAGGSLRL REREFLS QYADSVKG NLQMNGLKPEDTA AYVTVSS SCAVSGSTFG VYYCAG VEGFBII09A09/ EVQLVESGGG SMA WYRQAPG RISSEGTTARFTISRDNSKNTV FSSRPNP WGAGTT 73 LVQPGGSLRL KHRELVA YVDSVKG YLQMNSLKAEDTAVTVSS SCVASGIRFM VYYCNT VEGFBII09Al2/ EVQLVESGGG TDDVG WFRQAPG VIRWSTGGTRFTLSRDNAKNTM RSRPLGAGAWYTG WGQGTQ 74 LVQAGGSLRL KEREFVA YTSDSVAGYLQMNSLKPEDTA ETRYDS VTVSS SCAASGRTFS VYYCAA VEGFBII09D05/ EVQLVESGGGRYGMG WFRQAPG AISEYDNVY RFTISRDNSKSTV SPTILLSTDEWYK WGRGTQ 75 LVQPGDSLRLKEREFVI TADSVRG YLQMNSLKSEDTA Y VTVSS SCAASGLSFS VYYCAA VEGFBII09F05/EVQLVESGGG TDDVG WFRQAPG VIRWSTGGT RFTLSRDNAKNTM RSRPLGAGAWYTG WGQGTQ 76LVQAGGSLRL KEREFVA YTSDSVKG YLQMNSLKPEDTA ETRYNY VTVSS SCAASGRTFS VYYCAAVEGFBII10C007/ EVQLVESGGG NYAMG WFRQVPG VITRSPSNT RFTISRDNAKNIVHYWNSDSYTYTDS WGQGTQ 77 LVQAGGSLSL REREFVA YYTDSVKG YLQMNSLKPEDTA RWYNYVTVSS SCAASARAFS  VYYCAA VEGFBII10E07/ EVQLVESGGG NYAMG WFRQAPGDISSSGINT RFTISRDNAKNTV SAWWYSQMARDNY WGQGTQ 78 LVQAGGSLRL KERVLVAYVADAVKG YLQMNSLKPEDTA RY VTVSS SCAASGRTFS VYYCAA VEGFBII10G04/EVQLVESGGG RYAMG WFRQAPG SINTSGKRT RFAVSRDNAKNTG DRFFGSDSNEPRA WGQGTQ 79LVQAGGSLRL KEREFVA SYADSMKG YLQMNSLKLEDTA YRY VTVSS SCAASGDTLS TYYCAAVEGFBII10G05/ EVQLVESGGG NYNMG WFRQAPG TIRHHGYDT RFTISRDNAKNTVKLFWDMDPKTGFS WGQGTQ 80 LVQAGESLRL KEREFVA YYAESVKG YLQMNSLKPEDTA SVTVSS SCVASGITFS LYSCAK VEGFBII11C08/ EVQLVESGGG SYGLG WFRQAPG AIGWSGSSTRFTVSVDNAKNTV KVRNFNSDWDLLT WGQGTQ 81 LVQAGGSLRL KEREFVA YYADSVKGYLKMNSLEPEDTA SYNY VTVSS SCAASGRTLS VYYCAA VEGFBII11C11/ EVQLVESGGGSYAIG WFRQAPG RISWSGANT RFTISRGNAKNTV QTTSKYDNYDARA WGQGTQ 82 LVQAGGSLMLREREFVA YYADSVKG YLQMNSLKPEDTA YGY VTVSS SCAASGRALS AYYCAA VEGFBII11D09/EEQLVESGGG SYAIG WFRQAPG RISWSGANT RFTISRGNAKNTV QTTSKYDNYDARA WGQGTQ 83LVQAGGSLML REREFVA YYADSVKG YLQMNSLKPEDTA YGY VTVSS SCAASGRALS AYYCAAVEGFBII11E04/ EVQLVESGGG SYAMG WFRQAPG TISQSGYST RFTISRDNAKNTVDPFYSYGSPSPYR WGQGTQ 84 LVQAGGSLRL KEREFVA YYADSVKG NLQMNSLKPEDTA YVTVSS SCAASGRTFS VYYCAA VEGFBII11E05/ EVQLVESGGG FSAMG WFRQAPG AFKWSGSTTRFTISTDNAKNIL DRFYTGRYYSSDE WGQGTQ 85 LVQPGGSLRL KEREFVA YYADYVKGFLQMNSLKPEDTA YDY VTVSS SCASSGRLFS IYYCAV VEGFBII11F10/ EVQLVESGGG ITVMAWFRQAPG AITWSAPSS RFTISRDNAKNTV DRFKGRSIVTRSD WGQGTQ 86 LVQAGGSLRLKEREFVA YYADSVKG YLQVNSLKPEDSA YRY VTVSS SCAASTSIYS IYYCAA VEGFBII11F12/EVQLVESGGG SLAMG WFRQVPG SISQSGITT RFTISRDSAKNTV SVFYSTALTRPVD WGQGTQ 87LVQSGGSLRL KDREFVA SYADSVKS YLQMNLLKPEDTA YRY VTVSS SCAASGRSFS VYYCATVEGFBII11G09/ EVQLVESGGG ITVMA WFRQAPG AITWSAPTT RFTISRDNAKNTVDRFKGRSIVTRSD WGQGTQ 88 LVQAGGSLRL KEREFVA YSADSVKG YLQMNSLKPEDSA YRYVTVSS SCAASTSIYS IYYCAA VEGFBII12A07/ EVQLVESGGG KYVMG WFRQAPG AITSRDGPTRFTISGDNTKNKI DEDLYHYSSYHFT WGQGTQ 89 LVQAGGSLRL NDREFVA YYADSVKGFLQMNSLMPEDTA RVDLYHY VTVSS SCSVTGRTFN VYYCAI VEGFBII12B01/ EVQLVESGGGSSWMY WVRQAPG RISPGGLFT RFSVSTDNANNTL GGAPNYTP RGRGTQ 90 LVQPGGSLRLKGLEWVS YYVDSVKG YLQMNSLKPEDTA VTVSS ACAASGFTLS LYSCAK VEGFBII12C04/EVQLVESGGG SDVMG WYRQAPG FIRSLGSTY RFTISRDNAANTV RFSGESY WGQGTP 91LVQPGGSLRL KQRELVA YAGSVKG YLQMNNLKPEDTA VTVSS SCAASGSIVR VYYCNAVEGFBII12E10/ EVQLVESGGG NYVMG WFRQAPG AITSTNGPT RFTISGDNTKNKVDEDLYHYSSYHYT WGQGTQ 92 LAQAGGSLRL NEREFVA YYADSVKG FLQMDSLRPEDTARVALYHY VTVSS SCTASGRTFN VYYCAI VEGFBII12G04/ EVQLVESGGG LYAMG WFRQAPGAITWSAGDT RFTISRDNARNTV RQWGGTYYYHGSY WGQGTQ 93 LVQSGDSLRL REREFVSQYADSVKG NLQMNGLKPEDTA AW VTVSS SCAVSGNTFG VYYCAG VEGFBII16C03/EVQLVESEGG TDDVG WFRQAPG VIRWSTGGT RFTLSRDNAKNTM RSRPLGAGAWYTG WGQGTQ 94LVQAGGSLRL KEREFVA YTSDSVKG YLQMNSLKPEDTA ENYYNY VTVSS SCAASGRTFS VYYCAAVEGFBII16F11/ EVQLVESGGG GYDMG WFRQAPG AITWSGGST RFTISRDNAKNTVGRIWRSRDYDSEK WGHGTQ 95 LVQAGGSLRL KEREFVT YSPDSVKG YLQMNNLTPEDTA YYDIVTVSS SCAASGRTSS VYYCAS VEGFBII36C08/ EVQLVESGGG AYDMG WFRQAPG VISWTNSMTRFTISRDNAKNTV DRRRTYSRWRFYT WGQGTQ 96 LVQAGGSLRL KEREFVA YYADSVKGYLQMNSLKPEDTA GVNDYDY VTVSS SCAASGRTFS VYYCAV VEGFBII37F09/ EVQLVESGGGAYDMG WFRQAPG VISWSGGMT RFTISRDNAKSTV DRRRAYSRWRYYT WGQGTQ 97 LVQTGGSLRLKEREFVA YYADSVQG YLQMNSPKPEDTA GVNDYEF VTVSS SCAASGRTFS VYYCAVVEGFBII38A06/ EVQLVESGGG AYDMG WFRQAPG VISWSGGMT RFTISRDNAKNTVDRRRLYSRWRYYT WGQGTQ 98 LVQAGGSLRL KEREFVA YYADSVKG YLQMNSLKPEDTAGVNDYDY VTVSS SCAASGRTFS VYYCAV VEGFBII39H11/ EVQLVESGGG AYDMG WFRQAPGVISWTGGMT RFTISRDKAKNTV DRRRTYSRWRYYT WGQGTQ 99 LVQAGGSLRL KEREFVAYYADSVKG SLQMNSLKPEDTA GVNEYEY VTVSS SCAASGRTFS VYYCAV VEGFBII41B06/EVQLVESGGG AYDMG WFRQAPG VISWTGDMT RFTISRDKAKNTV DRRRTYSRWRYYT WGQGTQ100 LVQAGGSLRL KEREFVA YYADSVKG SLQMNSLKPEDTA GVNEYEY VTVSS SCAASGRTFSVYYCAA VEGFBII41C05/ EVQLVESGGG VYTMG WFRQAPG TISRTGDRT RFTISRENAKNTVGPIAPSPRPREYY WGQGTQ 101 LVQAGGSLRL KEREFVA SYANSVKG YLQMNSLKPEDTA YVTVSS SCAASGRTFS VYSCAA VEGFBII41D11/ EVQLMESGGG AYDMG WFRQAPG VISWTGGMTRFTISRDKAKNTV DRRRTYSRWRYYT WGQGTQ 102 LVQAGGSLRL KEREFVA YYADSVKGSLQMNSLKPEDTA GVNEYEY VTVSS SCAASGRTFS VYYCAV VEGFBII42F10/ EVQLVESGGGAYDMG WFRQAPG VISWSGGMT RFTISRENAKNTQ GRRRAYSRWRYYT WGQGTQ 103LVQAGGSLRL KEREFVA DYADSVKG FLQMNSLKPEDTA GVNEYDY VTVSS SCAASGRTFSVYYCAV VEGFBII86C11/ EVQLVESGGG SYAMG WFRQAPG HINRSGSST  RFTISRDNAKNTVGRYYSSDGVPSAS WGQGTQ 104 LVQAGDSLRL KERESVA YYADSVKG YLQLNSLKPEDTA FNYVTVSS SCTASGRTFN VYYCAA VEGFBII86F11/ EVQLVESGGG TWAMA WFRQAPG AISWSGSMTRFIISRDNAQNTL KTVDYCSAYECYA WGRGAQ 105 LVQAGDSLRL KEREFIS YYTDSVKGFLQMNNTAPEDTA RLEYDY VTVSS SCFTSARTFD VYYCAA VEGFBII86G08/ EVQLVESGGGSTNMG WFRQGPG AITLSGTTY RFTISRDNDKNTV DPSYYSTSRYTKA WGQGTQ 106LMQTGDSLRL KEREFVA YAEAVKG ALQMNSLKPEDTA TEYDY VTVSS SCAASGLRFT VYYCGAVEGFBII86G10/ EVQLVESGGG TYTMG WFRQTPG AIRWTVNIT RFTISRDIVKNTVQTSAPRSLIRMSN WGQGTQ 107 LVQAGGSLRL TEREFVA YYADSVKG YLQMNSLKPEDTA EYPYVTVSS SCAASGRTFN VYYCAA VEGFBII86G11/ EVQLVESGGG LYTVG WFRQAPG YISRSGSNRRFTLSRDNAKNTV TSRGLSSLAGEYN WGRGTQ 108 LVQAGGSLRL KEREFVA YYVDSVKGDLQMNSLKTEDTA Y VTVSS SCAASGLTFS VYYCAA VEGFBII86H09/ EVQLVESGGG SYRMGWFRRTPG SISWTYGST RFTMSRDKAKNAG GAQSDRYNIRSYD WGQGTQ 109 LVQAGGSLRLKEDEFVA FYADSVKG YLQMNSLKPEDTA Y VTVSS SCTASGSAFK LYYCAA VEGFBII87B07/EVQLVESGGG TSWMH WVRQAPG SIPPVGHFA RFTISRDNAKNTL DSAGRT KGQGTQ 110LVQPGGSLKL KGLEWVS NYAPSVKG FLQMNSLKSEDTA VTVSS SCTASGFTFS VYYCAKVEGFBII88A01/ KVQLVESGGG NYAMD WFRQAPG AITRSGGGT RFTISRDNAKNTV  TRSSTIVVGVGGM WGKGTL 111 LVQAGGSLRL KEREFVA YYADSVKG YLQMNSLKPEDTA EYVTVSS SCAASERTFS VYYCAA VEGFBII88A02/ EVQLVESGGG DYDIG WFRQAPG CITTDVGTTRFTISSDNAKNTV DTQDLGLDIFCRG WGQGTQ 112 LVQAGGSLRL NEREGVS YYADSVKGYLQINDLKPEDTA NGPFDG VTVSS SCAASGFTFG IYYCAV VEGFBII88B02/ EVQLVESGGGDYAIG WFRQAPG CISSYDSVT RFTISRDSAKNTL EREQLRRRESPHD SGKGTL 113LVQPGGSLRL KEREGVS YYADHVKG YLQMNSLSIEDTG ELLRLCFYGMRY VTVSS SCTASGLNLDVYYCAA VEGFBII88E02/ EVQLVESGGG DYAIG WFRQAPG CISSSDTSI RFTFSRDNAKNTVAFRCSGYELRGFP WGQGTQ 114 LVQPGGSLRL KEREAVS DYTNSVKG YLQMNSLKPEDTA TVTVSS SCVASGFRLD VYYCAA VEGFBII88G03/ EVQLVESGGG SLAVG WFRQAPG RITWSGATTRFTISRDNAKNTM DRSPNIINVVTAY WGQGTQ 115 LVQAGGSLRL KEREFVA YYADAVKDYLQMNSLKPEDTA EYDY VTVSS SCAASGGTFS VYYCAA VEGFBII88G05/ EVQLVESGGGLYNMG WFRQAPG AITSSPMST RFSISINNDKTTG PEGSFRRQYADRA WGQGTQ 116LVQPGASLRL KEREFVA YYADSVKG FLQMNVLKPEDTG MYDY VTVSS SCAASGDGFT VYFCAAVEGFBII88G11/ EVQLVESGGG GSDMG WFRQAPG AIRLSGSIT RFTISRDNAKNTVRSTYSYYLALADR WGQGTQ 117 LAQAGGSLRL KEREIVA YYPDSVKG YLQMNSLKPEDTA GGYDYVTVSS SCAASGRTFS VYYCAA VEGFBII88H01/ EVQLVESGGG TYAIG WFRQAPG CMSAGDSIPRFTTSTDNARNTV ARYHGDYCYYEGY WGQGTQ 118 LVQAGGSLRL KEREAVS WYTASVKGYLQMNSLKPEDTA YPF VTVSS SCVASGFTLG HYYCAA VEGFBII89B04/ EVQLVESGGG TNFMGWYRQAPG TITSSSITN RFTISRDNAKNTV RWRWSDVEY WGKGTL 119 LVQAGGSLRL KQRELVAYVDSVKG YLQMTSLKPEDTA VTVSS SCAASTSISS VYYCHA VEGFBII89B08/ EVQLVESGGGIFAMR WYRQAPG SITRSSITT RFTPSRDNAKNTV AIRPELYSVVNDY WGQGTQ 120LVQPGGSLRL KQRELVA YADSVKG SLQMNSLKPEDTA VTVSS SCAASGTTSS VYYCNAVEGFBII89D04/ EVQLVESGGG DYNLG WFRQAPG VISWRDSFA RFTISRDNAKNTVDRVSSRLVLPNTS WGQGTQ 121 LVQPGGSLRL KERQFVA YYAEPVKG YLQMNSLKPEDTA PDFGSVTVSS SCATSGLTFS VYYCAA VEGFBII89F09/ EVQLVESGGG NAIMG WFRQAPG AMNWRGGPTRFTISGDNTKNTV DEDLYHYSSYHYS WGQGTQ 122 LVQAGDSLRL QEREFVA YYADSVKGFLQMNFLKPEDTA RVDLYHY VTVSS SCAASGRTFN VYYCAA VEGFBII89G09/ EVQLVESGGGIFAMR WYRQAPG SITRSSITT RFTLSRDNAKNTV AIRPELYSVVNDY WGQGTQ 123LVQPGGSLRL KQRELVA YADSVKG SLQMNSLKPEDTA VTVSS SCAASGTTSS VYYCNAVEGFBII89H08/ EVQLVESGGG SYAPG WFRQAPG AFTRSSNIP RFTISRDNAHTVYNLGSTWSRDQRTY WGQGTQ 124 LVQAGGSLRL KEREFVA YYKDSVKG LQMNSLKPEDTAI DYVTVSS SCAASGGSFS YYCAV

TABLE 3 Sequence IDs and AA sequences of monovalent receptor-blocking anti-VEGF VHHs(FR, framework; CDR, complementary determining region) SEQ ID NO: 9-46VHH ID/ SEQ ID NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 VEGFBII22A10/EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 9GLVQPGDSL G REFVV YDSVSLEG VYLQTPSLKPED ADTYDY TVSS KLSCAFSGR TADYYCAATFS VEGFBII22A11/ EVQLVESGG SYSM WFRQAQGKE AISSGGFIY RFTISRDNTKNTSRAYGSSRLRL WGQGTQV 10 GLVQPGDSL A REFVV DAVSLEG VYLQTPSLKPED ADTYDYTVSS KLSCAFSGR TAVYYCAA TFS VEGFBII22B06/ EVQLVESGG SYSM WFRQAQGKEAISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 11 GLVQPGDSL G REFVV YDSVSLEGVYLQTPSLKPED ADTYDY TVSS KLSCAASGR TAVYYCAA TFS VEGFBII22B07/ EVQLVESGGSYSM WFRQAQGKE AISSSGNYK RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 12 GLVQAGDSL GREFVV YDSVSLEG VYLQINSLKPED GDTYDY TVSS RLSCAASGR TAVYYCAA TFSVEGFBII22E04/ EVQLVESGG SYSM WFRQAQGKE AISSGGSIY RFTISRDNTKNTSRAYASSRLRL WGQGTQV 13 GLVQPGDSL G REFVV DSVSLQG VYLQTPSLKPED ADTYDYTVSS KLSCVASGR TAVYYCAA TSS VEGFBII23A03/ EVQLVESGG SYSM WFRQAQGKEAISSGGYIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 14 GLVQPGDSL G REFVV DSVSLQGVYLQTPSLKPED ADTYDY TVSS KLSCVASGR TAVYYCAA TFS VEGFBII23A06/ EVQLVESGGSYSM WFRQAQGKE AISSGGFIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 15 GLVQPGDSL GREFVV DAVSLEG VYLQTPSLKPED ADTYDY TVSS KLSCAFSGR TAVYYCAA TFSVEGFBII23A08/ EVQLVESGG SYSM WFRQAQGKE AISNGGYKY RFTISRDNTKNTSRAYGSSRLRL WGQGTQV 16 GLVQTGDSL G REFVV DSVSLEG VYLQINSLKPED ADTYDYTVSS RLSCVASGR TAVYYCAA TFS VEGFBII23A09/ EVQLVESGG SYSM WFRQAQGKEAISSSGGYI RFTISRDNSKNT SRAYGSSRLRL WGQGTQV 17 GLVQPGDSL G REFVV YDSVSLEGVYLQTPSLKPED PDTYDY TVSS KLSCAFSGR TAVYYCAA TFG VEGFBII23B04/ EVQLVESGGSYSM WFRQAQGKE AISKGGYKY RFTISKDNAKNT SRAYGSSRLRL WGQGTQV 18 GLVQTGDSL GREFVV DSVSLEG VYLQINSLKPED ADTYEY TVSS RLSCEVSGR TAVYYCAS TFSVEGFBII23D11/ EVQLVESGG SYSM WFRQAQGKE AISSGGFIY RFTISRDNTKNTSRAYGSSRLRL WGQGTQV 19 GLVQPGDSL A REFVV DAVSLEG VYLQTPSLKPED ADTYDYTVSS RLSCAFSGR TAVYYCAA TFS VEGFBII23E05/ EVQLVESEG SYSM WFRQAQGKEAISSGGYIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 20 GLVQPGDSL G REFVV DSVSLQGVYLQTPSLKPED ADTYDY TVSS KLSCVASGR TAVYYCAA TSS VEGFBII23F02/ EMQLVESGGSYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 21 GLVQPGDSL GREFVV YDSVSLEG VYLQTPSLKPED ADTYDY TVSS KLSCAFSGR TADYYCAA TFSVEGFBII23F05/ EVQLVESGG SYSM WFRQAQGKE AISSSGNYK RFTISRDNTKNTSRAYGSSRLRL WGQGTQV 22 GLVQAGDSL G REFVV YDSVSLEG VYLQINSLKPKD GDTYDYTVSS RLSCAASGR TAVYYCAA TFS VEGFBII23F11/ EVQLVESGG SYSM WFRQAQGKEAISSGGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 23 GLVQPGDSL G REFVV YDSVSLEGVYLQTPSLKPED ADTYDY TVSS KLSCAFSGR TADYYCAA TFS VEGFBII23G03/ EVQLVESGGSYSM WFRQAQGKE AISSSGGYI RFTISRDNSKNT SRAYGSSRLRL WGQGTQV 24  GLVQPGDSLG REFVV YDSVSLEG VYLQTPSLKPED PGTYDY TVSS KLSCAFSGR TAVYYCAA TFGVEGFBII24C04/ EVQLVESGG SYSM WFRQAQGKE AISSGGYIY RFTISRDNTKNTSRAYGSSRLRL WGQGTQV 25 GLVQPGDSL G REFVV DSVSLQG VYLQTPSLKPED ADTYDYTVSS KLSCVASGR TAVYYCAA TSS VEGFBII27D08/ EVQLVESGG SYSM WFRQAQGKEAISSGGYKY RFTISRDNTQNT SRAYGSGRLRL WGQGTQV 26 GLVQTGDSL G REFVV DSVSLEGVYLQINSLKPED ADTYDY TVSS RLSCAASGR TAVYYCAA TFS VEGFBII27G07/ EVQLVESGGSYSM WFRQAQGQE AISSGGYIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 27 GLVQPGDSL GREFVV DSVSLQG VYLQTPSLKPED ADTYDY TVSS KLSCVASGR TAVYYCAA TSSVEGFBII30C09/ EVQLVESGG SYSM WFRQAQGQE AISSGGYIY RFTISRDNTKNTSRAYGSSRLRL WGQGTQV 28 GLVQPGDSL G REFVV DSVSLQG VYLQTPSLKPED ADTYDYTVSS KLSCIASGR TAVYYCAA TSS VEGFBII30E07/ EVQLVESGG SYSM WFRQAQGKEAISSSGNYK RFTISRDNTKNT SRAYGSSRLRL WGQGTRV 29 GLVQAGDSL G REFVV YDSVSLEGVYLQINSLKPED GDTYDY TVSS RLSCAASGR TAVYYCAA TFS VEGFBII31C07/ EVQLVESGGSYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 30 GLVQTGDSL GREFVV YDSVSLEG VYLQTPSLKPED ADTYDY TVSS RLSCAASGG TADYYCAA TFSVEGFBII39E02/ EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNTSRAYGSSRLRL WGQGTQV 31 GLVQPGDPL G REFVV YDSVSLEG VYLQTPSLKPED ADTYDYTVSS KLSCAFSGR TADYYCAA TFS VEGFBII39G04/ EVPLVESGG SYSM WFRQAQGKEAISSSGNYK RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 32 GLVQAGDSL G REFVV YDSASLEGVYLQINSLKPED GDTYDY TVSS RLSCAASGR TAVYYCAA TFS VEGFBII40F02/ EVQLVESGGSYSM WFRQAQGKE AISSGGFIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 33 GLVQPGDSL AREFVV DAVSLEG VYLQTPSLKPEG ADTYDY TVSS KLSCAFSGR TAVYYCAA TFSVEGFBII40G07/ EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNASRAYGSSRLRL WGQGTQV 34 GLVQPGDSL G REFVV YDSVSLEG VYLQTPSLKPED ADTYDYTVSS KLSCAFSGR TADYYCAA TFS VEGFBII40H10/ EVQLMESGG SYSM WFRQAQGKEAISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 35 GLVQPGDSL G REFVV YDSVSLEGVYLQTPSLKPED ADTYDY TVSS KLSCAFSGR TADYYCAA TFS VEGFBII41B05/ EVQLVESGGSYSM WFRQAQGKE AISSGGFIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 36 GLVQPGGSL GREFVV DAVSLEG VYLQTPSLKPED ADTYDY TVSS RLSCAFSGR TAVYYCAA TFSVEGFBII41G03/ EVQLVESGG SYSM WFRQAQGKE AISSGGFIY RFTISRENTKNTSRAYGSSRLRL WGQGTQV 37 GLVQPGDSL A REFVV DAVSLEG VYLQTPSLKPED ADTYDYTVSS KLSCAFSGR TAVYYCAA TFS VEGFBII42A05/ EVQLVESGG SYSM WFRQAQGKEAISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 38 GLVQPGDSL G REFVV YDSVSLEGVYLQTPSLKPED ADTYDY TVSS KLSCAFSGR TADYYCAA TFS VEGFBII42D05/ EVQLVESGGSYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 39 GLVQPGDSL GREFVV YDSVSLEG VYLQTPSLKPED ADTYDY TVSS KLSCAFSGR TAVYYCAA TFSVEGFBII42F11/ EVQLVESGG SYSV WFRQAQGKE AISSGGYIY RFTISRDNTKNTSRAYGSSRLRL WGQGTQV 40 GLVQPGDSL G REFVV DSVSLQG VYLQTPSLKPED ADTYDYTVSS KLSCVASGR TAVYYCAA TSS VEGFBII56E11/ EVQLVESGG SYSM WFRQAQGKEAISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 41 GLVQPGDSL G REFVV YDSVSLEGVYLQTPSLKPED ADTYDY TVSS KLSCAFSGR AADYYCAA TFS VEGFBII60A09/ EVQLVESGGSYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 42 GLVQPGDSL GREFVV YDSVSLEG VYLQTPSLKPED ADTYDY TVSS KLSCAFSGR TADYYCAA TFSVEGFBII61A01/ EVQLVESGG SYSM WFRQAQGKE AISSGGYKY RFTISRDNTKNTSRAYASSRLRL WGQGTQV 43 GLVQAGGSL G REFVV DAVSLEG VYLQTPSLKPED ADTYDYTVSS RLSCAFSGR TAVYYCAA TFS VEGFBII62A09/ EVQLVESGG SYSM WFRQAQGKEAISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 44 DLVQPGDSL G REFVV YDSVSLEGVYLQTPSLKPED ADTYDY TVSS KLSCAASGR TAVYYCAA TFS VEGFBII62D10/ EVQLVESEGSYSM WFRQAQGKE AISSSGNYK RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 45 GLVQAGDSL GREFVV YDSVSLEG VYLQINSLKPED GDTYDY TVSS RLSCAASGR TAVYYCAA TFSVEGFBII62F02/ EVQLVESGG SYSM WFRQAQGKE AIASGGYIY RFTISRDNTKDTSRAYGSSRLRL WGQGTQV 46 GLVQPGDSL G REFVV DAVSLEG VYLQTPSLKPED ADTYDYTVSS KLSCAFSGR TAVYYCAA TFS

Dissociation rates of inhibitory VHHs are analyzed on Biacore (BiacoreT100 instrument, GE Healthcare). HBS-EP+ buffer is used as runningbuffer and experiments are performed at 25° C. Recombinant human VEGF165is irreversibly captured on a CM5 sensor chip via amine coupling (usingEDC and NHS) up to a target level of +/−1500RU. After immobilization,surfaces are deactivated with 10 min injection of 1M ethanolamine pH8.5. A reference surface is activated and deactivated with respectivelyEDC/NHS and ethanolamine. Periplasmic extracts of VHHs are injected at a10-fold dilution in running buffer for 2 min at 45 μl/min and allowed todissociate for 10 or 15 min. Between different samples, the surfaces areregenerated with regeneration buffer. Data are double referenced bysubtraction of the curves on the reference channel and of a blankrunning buffer injection. The of the processed curves is evaluated byfitting a two phase decay model in the Biacore T100 Evaluation softwarev2.0.1. Values for k_(d)-fast, k_(d)-slow and % fast are listed in Table4.

TABLE 4 Off-rate determination of anti-VEGF receptor-blocking VHHs withBiacore B- Unique Bind- cell se- Represen- ing line- quence tative k_(d)k_(d) % level age variant VHH ID (fast) (slow) fast (RU) 1 1VEGFBII22B07 1.50E−02 7.80E−05 31 328 1 2 VEGFBII23A08 1.30E−02 5.00E−0519 502 1 3 VEGFBII23B04 8.80E−03 4.00E−05 12 768 1 4 VEGFBII27D082.40E−02 8.10E−05 13 225 1 5 VEGFBII24C04 1.30E−02 3.40E−05 17 456 1 6VEGFBII27G07 1.30E−02 3.80E−05 18 471 1 7 VEGFBII22E04 1.80E−02 1.10E−0414 520 1 8 VEGFBII23A03 1.50E−02 3.20E−05 15 487 1 9 VEGFBII22B063.80E−02 9.00E−05 23 168 1 10 VEGFBII23A09 2.70E−02 4.60E−05 20 247 1 11VEGFBII23G03 2.80E−02 8.60E−05 28 141 1 12 VEGFBII22A11 2.20E−024.70E−05 12 461 1 13 VEGFBII23A06 1.70E−02 3.70E−05 13 547 1 14VEGFBII23F11 2.70E−02 1.30E−04 22 134 1 15 VEGFBII22A10 3.70E−024.00E−05 19 229 1 16 VEGFBII23F05 1.60E−02 1.30E−04 29 198 1 17VEGFBII23D11 1.90E−02 5.80E−05 13 510 1 18 VEGFBII23F02 n/d n/d n/d n/d1 19 VEGFBII23E05 1.50E−02 6.90E−05 18 275 1 20 VEGFBII31C07 3.70E−021.50E−04 25 77 1 21 VEGFBII30C09 1.50E−02 7.60E−05 19 264 1 22VEGFBII30E07 1.70E−02 1.30E−04 29 226 1 23 VEGFBII39G04 1.40E−027.40E−04 40 210 1 24 VEGFBII41G03 1.20E−02 2.70E−04 20 332 1 25VEGFBII41B05 1.90E−02 1.20E−04 16 324 1 26 VEGFBII40F02 1.20E−029.80E−05 20 258 1 27 VEGFBII39E02 1.90E−02 2.40E−04 13 181 1 28VEGFBII42D05 3.30E−02 1.50E−04 26 77 1 29 VEGFBII40G07 1.80E−02 3.20E−0419 139 1 30 VEGFBII42A05 1.60E−02 3.40E−04 25 118 1 31 VEGFBII42F119.10E−03 5.00E−04 46 100 1 32 VEGFBII40H10 1.40E−02 2.90E−04 17 200 1 33VEGFBII62A09 4.10E−02 1.10E−04 23 84 1 34 VEGFBII60A09 3.70E−02 9.30E−0520 106 1 35 VEGFBII62F02 1.40E−02 8.50E−05 21 205 1 36 VEGFBII62D101.90E−02 1.60E−04 40 94 1 37 VEGFBII61A01 7.40E−03 1.70E−04 21 275 1 38VEGFBII56E11 3.30E−02 1.40E−04 24 76 n/d, not determined

Example 5 Characterization of Purified Anti-VEGF VHHs

Three inhibitory anti-VEGF VHHs are selected for furthercharacterization as purified protein: VEGFBII23B04, VEGFBII24C4 andVEGFBII23A6. These VHHs are expressed in E. coli TG1 as c-myc,His6-tagged proteins. Expression is induced by addition of 1 mM IPTG andallowed to continue for 4 hours at 37° C. After spinning the cellcultures, periplasmic extracts are prepared by freeze-thawing thepellets. These extracts are used as starting material for VHHpurification via IMAC and size exclusion chromatography (SEC). Final VHHpreparations show 95% purity as assessed via SDS-PAGE.

5.1 Evaluation of Human VEGF165/VEGFR2 Blocking VHHs in HumanVEGF165/Human VEGFR2-Fc Blocking ELISA

The blocking capacity of the VHHs is evaluated in a human VEGF165/humanVEGFR2-Fc blocking ELISA. In brief, 1 μg/mL of VEGFR2-Fc chimera (R&DSystems, Minneapolis, Minn., USA) is coated in a 96-well MaxiSorp plate(Nunc; Wiesbaden, Germany). Dilution series (concentration range 1 mM-64pM) of the purified VHHs in PBS buffer containing 0.1% casein and 0.05%Tween 20 (Sigma) are incubated in the presence of 4 nM biotinlyatedVEGF165. Residual binding of bio-VEGF165 to VEGFR2 is detected usinghorseradish peroxidase (HRP) conjugated extravidin (Sigma, St Louis,Mo., USA) and TMB as substrate. As controls Bevacizumab (Avastin®) andRanibizumab (Lucentis®) are taken along. Dose inhibition curves areshown in FIG. 1; the corresponding IC₅₀ values and % inhibition aresummarized in Table 5.

TABLE 5 IC₅₀ (nM) values and % inhibition for monovalent VHHs inhVEGF165/hVEGFR2-Fc competition ELISA IC₅₀ % VHH ID (nM) inhibitionVEGFBII23B04 2.1 100 VEGFBII23A06 3.0 100 VEGFBII24C04 2.5 100Ranibizumab 1.6 100 Bevacizumab 1.7 100

5.2 Evaluation of Human VEGF165/VEGFR2 Blocking VHHs in HumanVEGF165/Human VEGFR 1-Fc Blocking ELISA

VHHs are also evaluated in a human VEGF165/human VEGFR1-Fc blockingELISA. In brief, 2 μg/mL of VEGFR1-Fc chimera (R&D Systems, Minneapolis,Minn., USA) is coated in a 96-well MaxiSorp plate (Nunc, Wiesbaden,Germany). Dilution series (concentration range 1 mM-64 pM) of thepurified VHHs in PBS buffer containing 0.1% casein and 0.05% Tween 20(Sigma) are incubated in the presence of 0.5 nM biotinlyated VEGF165.Residual binding of bio-VEGF165 to VEGFR1 is detected using horseradishperoxidase (HRP) conjugated extravidin (Sigma, St Louis, Mo., USA) andTMB as substrate. As controls Bevacizumab, Ranibizumab and an irrelevantVHH (2E6) are taken along. Dose inhibition curves are shown in FIG. 2;the corresponding IC₅₀ values and % inhibition are summarized in Table6.

TABLE 6 IC₅₀ (nM) values and % inhibition of monovalent VHHs inhVEGF165/hVEGFR1-Fc competition ELISA % VHH ID IC₅₀ (nM) inhibitionVEGFBII23B04 0.5 64 VEGFBII23A06 0.9 55 VEGFBII24C04 0.8 71 Ranibizumab1.2 91 Bevacizumab 1.5 965.3 Evaluation of the Anti-VEGF165 VHHs in the Human VEGF165/humanVEGFR2-Fc Blocking AlphaScreen

The blocking capacity of the VHHs is also evaluated in a humanVEGF165/human VEGFR2-Fc blocking AlphaScreen. Briefly, serial dilutionsof purified VHHs (concentration range: 200 nM-0.7 pM) in PBS buffercontaining 0.03% Tween 20 (Sigma) are added to 4 pM bio-VEGF165 andincubated for 15 min. Subsequently VEGFR2-Fc (0.4 nM) and anti-FcVHH-coated acceptor beads (20 μg/ml) are added and this mixture isincubated for 1 hour in the dark. Finally, streptavidin donor beads (20μg/ml) are added and after 1 hour of incubation in the dark,fluorescence is measured on the Envision microplate reader.Dose-response curves are shown in the FIG. 3. The IC₅₀ values for VHHsblocking the human VEGF165-human VEGFR2-Fc interaction are summarized inTable 7.

TABLE 7 IC₅₀ (pM) values and % inhibition for VHHs inhVEGF165/hVEGFR2-Fc competition AlphaScreen % VHH ID IC₅₀ (pM)inhibition VEGFBII23B04 160 100 VEGFBII23A06 250 100 VEGFBII24C04 250100 Ranibizumab 860 1005.4 Evaluation of the Anti-VEGF165 VHHs in the Human VEGF165/humanVEGFR1-Fc Blocking AlphaScreen

The blocking capacity of the VHHs is also evaluated in a humanVEGF165/human VEGFR1-Fc blocking AlphaScreen. Briefly, serial dilutionsof purified VHHs (concentration range: 500 nM-1.8 pM)) in PBS buffercontaining 0.03% Tween 20 (Sigma) are added to 0.4 nM bio-VEGF165 andincubated for 15 min. Subsequently VEGFR1-Fc (1 nM) and anti-FcVHH-coated acceptor beads (20 μg/ml) are added and this mixture isincubated for 1 hour in the dark. Finally, streptavidin donor beads (20μg/ml) are added and after 1 hour of incubation in the dark,fluorescence is measured on the Envision microplate reader.Dose-response curves are shown in the FIG. 4. The IC₅₀ values and %inhibition for VHHs blocking the human VEGF165-human VEGFR1-Fcinteraction are summarized in Table 8.

TABLE 8 IC₅₀ (nM) values for VHHs in hVEGF165/hVEGFR1-Fc competitionAlphaScreen VHH ID IC₅₀ (nM) % inhibition VEGFBII23B04 0.9 41VEGFBII23A06 0.4 46 VEGFBII24C04 0.2 53 Ranibizumab 3.3 79

5.5 Determination of the Affinity of the Human VEGF165-VHH Interaction

Binding kinetics of VHH VEGFBII23B04 with hVEGF165 is analyzed by SPR ona Biacore T100 instrument. Recombinant human VEGF165 is immobilizeddirectly on a CM5 chip via amine coupling (using EDC and NHS). VHHs areanalyzed at different concentrations between 10 and 360 nM. Samples areinjected for 2 min and allowed to dissociate up to 20 min at a flow rateof 45 μl/min. In between sample injections, the chip surface isregenerated with 100 mM HCl. HBS-EP+(Hepes buffer pH 7.4+EDTA) is usedas running buffer. Binding curves are fitted using a Two State Reactionmodel by Biacore T100 Evaluation Software v2.0.1. The calculatedaffinities of the anti-VEGF VHHs are listed in Table 9.

TABLE 9 Affinity K_(D) (nM) of purified VHHs for recombinant humanVEGF165 VEGF165 k_(a) k_(a1) k_(a2) k_(d) k_(d1) k_(d2) K_(D) VHH ID(M⁻¹ · s⁻¹) (M⁻¹ · s⁻¹) (M⁻¹ · s⁻¹) (s⁻¹) (s⁻¹) (s⁻¹) (nM)VEGFBII23B04^((a)) — 2.1E+05 1.4E−02 — 8.6E−03 2.4E−04 0.7VEGFBII23A06^((a)) — 4.2E+05 2.0E−02 — 5.7E−02 1.0E−04 0.7VEGFBII24C04^((a)) — 3.2E+05 1.8E−02 — 2.6E−02 9.6E−05 0.4^((a))Heterogeneous binding curve resulting in no 1:1 fit, curves arefitted using a Two State Reaction model by Biacore T100 EvaluationSoftware v2.0.1

5.6 Binding to Mouse VEGF164

Cross-reactivity to mouse VEGF164 is determined using a binding ELISA.In brief, recombinant mouse VEGF164 (R&D Systems, Minneapolis, Miss.,USA) is coated overnight at 4° C. at 1 μg/mL in a 96-well MaxiSorp plate(Nunc, Wiesbaden, Germany). Wells are blocked with a casein solution (1%in PBS). VHHs are applied as dilution series (concentration range: 500nM-32 pM) in PBS buffer containing 0.1% casein and 0.05% Tween 20(Sigma) and binding is detected using a mouse anti-myc (Roche) and ananti-mouse-HRP conjugate (DAKO) and a subsequent enzymatic reaction inthe presence of the substrate TMB (3,3′,5,5′-tetramentylbenzidine)(Pierce, Rockford, Ill., USA) (FIG. 5). A mouse VEGF164 reactive mAb isincluded as positive control. As reference, binding to human VEGF165 isalso measured. EC₅₀ values are summarized in Table 10.

TABLE 10 EC₅₀ (pM) values for VHHs in a recombinant human VEGF165 andmouse VEGF164 binding ELISA rhVEGF165 rmVEGF164 VHH ID EC₅₀ (pM) EC₅₀(pM) VEGFBII23B04 297 NB VEGFBII24C04 453 NB VEGFBII23A06 531 NB NB, nobinding

5.7 Binding to VEGF121

Binding to recombinant human VEGF121 is assessed via a solid phasebinding ELISA. Briefly, recombinant human VEGF121 (R&D Systems,Minneapolis, Miss., USA) is coated overnight at 4° C. at 1 μg/mL in a96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blockedwith a casein solution (1% in PBS). VHHs are applied as dilution series(concentration range: 500 nM-32 pM) in PBS buffer containing 0.1% caseinand 0.05% Tween 20 (Sigma) and binding is detected using a mouseanti-myc (Roche) and an anti-mouse-HRP conjugate (DAKO) and a subsequentenzymatic reaction in the presence of the substrate TMB(3,3′,5,5′-tetramentylbenzidine) (Pierce, Rockford, Ill., USA) (FIG. 6).As positive control serial dilutions of the VEGFR2 is taken along. EC₅₀values are summarized in Table 11.

TABLE 11 EC₅₀ (pM) values for monovalent VHHs in a recombinant humanVEGF121 binding ELISA VHH ID EC₅₀ (pM) VEGFBII23B04 510 VEGFBII24C04 792VEGFBII23A06 928

5.8 Binding to VEGF Family Members VEGFB, VEGFC, VEGFD and PIGF

Binding to VEGFB, VEGFC, VEGFD and PIGF is assessed via a solid phasebinding ELISA. In brief, VEGFB, VEGFC, VEGFD and PIGF (R&D Systems,Minneapolis, Miss., USA) are coated overnight at 4° C. at 1 μg/mL in a96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blockedwith a casein solution (1% in PBS). VHHs are applied as dilution series(concentration range: 500 nM-32 pM) and binding is detected using amouse anti-myc (Roche) and an anti-mouse-AP conjugate (Sigma, St Louis,Mo., USA). As positive controls serial dilutions of the appropriatereceptors are taken along and detected with horseradish peroxidase(HRP)-conjugated goat anti-human IgG, Fc specific antibody (JacksonImmuno Research Laboratories Inc., West Grove, Pa., USA) and asubsequent enzymatic reaction in the presence of the substrate TMB(3,3′,5,5′-tetramentylbenzidine) (Pierce, Rockford, Ill., USA).Dose-response curves of VHHs and controls are shown in FIG. 7. Theresults show that there was no detectable binding of the selected VHHsto VEGFB, VEGFC, VEGFD or PIGF.

5.9 Epitope Binning

Biacore-based epitope binning experiments are performed to investigatewhich VEGF binders bind to a similar or overlapping epitope asVEGFBII23B04. To this end, VEGFBII23B04 is immobilized on a CM5 sensorchip. For each sample, human VEGF165 is passed over the chip surface andreversibly captured by VEGFBII23B4. Purified VHHs (100 nM) orperiplasmic extracts (1/10 diluted) are then injected with a surfacecontact time of 240 seconds and a flow rate of 10 uL/minute. Betweendifferent samples, the surface is regenerated with regeneration buffer(100 mM HCl). Processed curves are evaluated with Biacore T100Evaluation software. VHHs could be divided within two groups: group onewhich gave additional binding to VEGFBII23B04 captured VEGF165 and asecond group which is not able to simultaneously bind to VEGFBII23B04captured VEGF165. Table 12-A summarizes the binding epitopes of thetested VHHs.

The same assay set-up is used to assess whether VEGFR1, VEGFR2,Ranibizumab and Bevacizumab are able to bind to human VEGF-165simultaneously with VEGFBII23B04. Table 12-B presents the additionalbinding responses to VEGFBII23B04-captured VEGF165. Only VEGFR2 is notable to bind to VEGFBII23B04-captured VEGF165, underscoring the blockingcapacity of VEGFBII23B04 for the VEGF-VEGFR2 interaction. In addition,these data show that the VEGFBII23B04 epitope is different from theBevacizumab and Ranibizumab epitope.

TABLE 12-A Epitope binning of anti-VEGF VHHs - simultaneous binding withVEGFBII23B04 No or low 1C02 1E07 4B08 8E07 8F07 12A07 12B01 86C11 86F1186G08 additional 86G10 86G11 87B07 88A01 88A02 88B02 88E02 88G03 88G0588G11 binding to 88H01 89B04 89D04 89F09 89G09 89H08 24C04 23A6 27G0723B04 23B04- captured VEGF165* Additional 3D12 5B02 5B03 5B05 6G02 7D088D09 8F06 10C07 10E07 binding to 10G04 10G05 11C08 11D09 11E04 11E0511F12 86H09 41C05 23B04- captured VEGF165 *indicating same oroverlapping epitopes

TABLE 12-B Epitope binning of VEGFBII23B04 - binding of benchmarkinhibitors or cognate receptors on VEGFBII23B04 captured VEGF165Injection Binding step Binding [sample] level (RU) 1 VEGF165 100 nM 17272 VEGFBII23B04 100 nM — 3 Ranibizumab 100 nM  763 4 Bevacizumab 100 nM1349 5 VEGFR1 100 nM 1011 6 VEGFR2 100 nM —

5.10 Characterization of the Anti-VEGF VHHs in the HUVEC ProliferationAssay

The potency of the selected VHHs is evaluated in a proliferation assay.In brief, primary HUVEC cells (Technoclone) are supplement-starved overnight and then 4000 cells/well are seeded in quadruplicate in 96-welltissue culture plates. Cells are stimulated in the absence or presenceof VHHs with 33 ng/mL VEGF. The proliferation rates are measured by[³H]Thymidine incorporation on day 4. The results of the HUVECproliferation assay are shown in Table.

TABLE 13 IC₅₀ (nM) values and % inhibition of monovalent VEGFBII23B04,VEGFBII23A06 and VEGFBII24C04 in VEGF HUVEC proliferation assay VHH IDIC₅₀ (nM) % inhibition VEGFBII23B04 0.36 91 Bevacizumab 0.21 92VEGFBII23A06 4.29 73 VEGFBII24C04 3.8 79 Bevacizumab 0.78 78

5.11 Characterization of the Anti-VEGF VHHs in the HUVEC ErkPhosphorylation Assay

The potency of the selected VHHs is assessed in the HUVEC Erkphosphorylation assay. In brief, primary HUVE cells are serum-starvedover night and then stimulated in the absence or presence of VHHs with10 ng/mL VEGF for 5 min. Cells are fixed with 4% Formaldehyde in PBS andERK phosphorylation levels are measured by ELISA usingphosphoERK-specific antibodies (anti-phosphoMAP Kinase pERK1&2, M8159,Sigma) and polyclonal Rabbit Anti-Mouse-Immunoglobulin-HRP conjugate(PO161, Dako). As shown in Table 14, VEGFBII23B04 and Bevacizumabinhibit the VEGF induced Erk phosphoryaltion by at least 90%, withIC₅₀s<1 nM.

TABLE 14 IC₅₀ (nM) values and % inhibition of monovalent VEGFBII23B04 inVEGF HUVEC Erk phosphorylation assay VHH ID IC₅₀ (nM) % inhibitionVEGFBII23B04 0.37 90 Bevacizumab 0.63 98

Example 6 Generation of Multivalent Anti-VEGF Blocking VHHs

VHH VEGFBII23B04 is genetically fused to either VEGFBII23B04 resultingin a homodimeric VHH (AA sequence see Table 15) or different VEGFbinding VHHs resulting in heterodimeric VHHs. To generate theheterodimeric VHHs, a panel of 10 unique VEGF binding VHHs are linkedvia a 9 or 40 Gly-Ser flexible linker in two different orientations toVEGFBII23B04 (AA sequences see Table 15). Homodimeric VEGFBII23B04(VEGFBII010) and the 40 heterodimeric bivalent VHHs are expressed in E.coli TG1 as c-myc, His6-tagged proteins. Expression is induced byaddition of 1 mM IPTG and allowed to continue for 4 hours at 37° C.After spinning the cell cultures, periplasmic extracts are prepared byfreeze-thawing the pellets. These extracts are used as starting materialand VHHs are purified via IMAC and desalting resulting in 90% purity asassessed via SDS-PAGE.

TABLE 15Sequence ID, VHH ID and AA sequence of bivalent anti-VEGF VHHs (each of the usedlinkers is highlighted in one relevant sequence) Sequence ID/ SEQ ID NO:VHH ID AA sequence VEGFBII23B04- VEGFBII010EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS35GS-23B04/128LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVS

RTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBII23B04-EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS9GS-4B08/129LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVS

PSGYTYYWDFVKGRFTISRDNSKNIVYLQMNSLKPEDTAAYYCNTQFYWGQGTQVTVSSVEGFBII23B04-EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS9GS-5B03/130LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLAQAGDSLRLSCAASGRSFSHYNMGWFRQAPGKEREFVASIRGGGGSTTYANSVKDRFTISRENAKNTVYLQMNSLKPEDTAVYYCAATAFYRGPYDYDYWGQG TQVTVSSVEGFBII23B04- VEGFBII022EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS9GS-5B05/131LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCVASGIRFMSMAWYRQAPGKHRELVARISSGGTTAYVDSVKGRFTISRDNSKNTVYLQMNSLKAEDTAVYYCNTFSSRPNPWGAGTQVTVSSVEGFBII23B04-EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS9GS-6G02/132LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGNIFSNNAMAWYRQAPGKQRELVARISSGGGFTYYLDSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCNAAYRTYNYWGQGTQVTV SSVEGFBII23B04-EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS9GS-10E07/133LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSNYAMGWFRQAPGKERVLVADISSSGINTYVADAVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASAWWYSQMARDNYRYWGQGTQVTVSS VEGFBII23B04-EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS9GS-12B01/134LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLACAASGFTLSSSWMYWVRQAPGKGLEWVSRISPGGLFTYYVDSVKGRFSVSTDNANNTLYLQMNSLKPEDTALYSCAKGGAPNYTPRGRGTQVT VSSVEGFBII23B04-EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS9GS-86C11/135LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGDSLRLSCTASGRTFNSYAMGWFRQAPGKERESVAHINRSGSSTYYADSVKGRFTISRDNAKNTVYLQLNSLKPEDTAVYYCAAGRYYSSDGVPSASFNYWGQGTQVTVSS VEGFBII23B04-EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS9GS-86H09/136LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCTASGSAFKSYRMGWFRRTPGKEDEFVASISWTYGSTFYADSVKGRFTMSRDKAKNAGYLQMNSLKPEDTALYYCAAGAQSDRYNIRSYDYWG QGTQVTVSSVEGFBII23B04-EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS9GS-87B07/137LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCTASGFTFSTSWMHWVRQAPGKGLEWVSSIPPVGHFANYAPSVKGRFTISRDNAKNTLFLQMNSLKSEDTAVYYCAKDSAGRTKGQGTQVTVS SVEGFBII23B04-EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS9GS-88A01/138LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASERTFSNYAMDWFRQAPGKEREFVAAITRSGGGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAATRSSTIVVGVGGMEYWGKGTQVTVSS VEGFBII23B04-EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS40GS-4B08/139LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVS

CAASGSAVGDITVAWYRQAPGIQRQLVATITPSGYTYYWDFVKGRFTISRDNSKNIVYLQMNSLKPEDTAAYYCNTQFYWGQGTQVTVSS VEGFBII23B04-EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS40GS-5B03/140LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLAQAGDSLRLSCAASGRSFSHYNMGWFRQAPGKEREFVASIRGGGGSTTYANSVKDRFTISRENAKNTVYLQMNSLKPEDTAVYYCAATAFYRGPYDYDYWGQGTQVTVSS VEGFBII23B04- VEGFBII021EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS40GS-5B05/141LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGIRFMSMAWYRQAPGKHRELVARISSGGTTAYVDSVKGRFTISRDNSKNTVYLQMNSLKAEDTAVYYCNTFSSRPNPWGAGTQVTVSS VEGFBII23B04-EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS40GS-6G02/142LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGNIFSNNAMAWYRQAPGKQRELVARISSGGGFTYYLDSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCNAAYRTYNYWGQGTQVTVSS VEGFBII23B04- VEGFBII023EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS40GS-10E07/143LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSNYAMGWFRQAPGKERVLVADISSSGINTYVADAVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASAWWYSQMARDNYRYWGQGTQVTVSS VEGFBII23B04-EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS40GS-12B01/144LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLACAASGFTLSSSWMYWVRQAPGKGLEWVSRISPGGLFTYYVDSVKGRFSVSTDNANNTLYLQMNSLKPEDTALYSCAKGGAPNYTPRGRGTQVTVSS VEGFBII23B04-EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS40GS-86C11/145LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGDSLRLSCTASGRTFNSYAMGWFRQAPGKERESVAHINRSGSSTYYADSVKGRFTISRDNAKNTVYLQLNSLKPEDTAVYYCAAGRYYSSDGVPSASFNYWGQGTQVTVSS VEGFBII23B04- VEGFBII024EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS40GS-86H09/146LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCTASGSAFKSYRMGWFRRTPGKEDEFVASISWTYGSTFYADSVKGRFTMSRDKAKNAGYLQMNSLKPEDTALYYCAAGAQSDRYNIRSYDYWGQGTQVTVSS VEGFBII23B04-EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS40GS-87B307/147LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCTASGFTFSTSWMHWVRQAPGKGLEWVSSIPPVGHFANYAPSVKGRFTISRDNAKNTLFLQMNSLKSEDTAVYYCAKDSAGRTKGQGTQVTVSS VEGFBII23B04-EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS40GS-88A01/148LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASERTFSNYAMDWFRQAPGKEREFVAAITRSGGGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAATRSSTIVVGVGGMEYWGKGTQVTVSS VEGFBII4B08-EVQLVESGGGLVQPGGSLRLSCAASGSAVGDITVAWYRQAPGIQRQLVATITPSGYTYYWDF9GS-23B04/149VKGRFTISRDNSKNIVYLQMNSLKPEDTAAYYCNTQFYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBII5B03-EVQLVESGGGLAQAGDSLRLSCAASGRSFSHYNMGWFRQAPGKEREFVASIRGGGGSTTYAN9GS-23B04/150SVKDRFTISRENAKNTVYLQMNSLKPEDTAVYYCAATAFYRGPYDYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQG TQVTVSSVEGFBII5B05-EVQLVESGGGLVQPGGSLRLSCVASGIRFMSMAWYRQAPGKHRELVARISSGGTTAYVDSVK9GS-23B04/151GRFTISRDNSKNTVYLQMNSLKAEDTAVYYCNTFSSRPNPWGAGTQVTVSSGGGGSGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSSVEGFBII6G02-EVQLVESGGGLVQPGGSLRLSCAASGNIFSNNAMAWYRQAPGKQRELVARISSGGGFTYYLD9GS-23B04/152SVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCNAAYRTYNYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTV SSVEGFBII10E07-EVQLVESGGGLVQAGGSLRLSCAASGRTFSNYAMGWFRQAPGKERVLVADISSSGINTYVAD9GS-23B04/153AVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASAWWYSQMARDNYRYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBII12B01-EVQLVESGGGLVQPGGSLRLACAASGFTLSSSWMYWVRQAPGKGLEWVSRISPGGLFTYYVD9GS-23B04/154SVKGRFSVSTDNANNTLYLQMNSLKPEDTALYSCAKGGAPNYTPRGRGTQVTVSSGGGGSGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVT VSSVEGFBII86C11-EVQLVESGGGLVQAGDSLRLSCTASGRTFNSYAMGWFRQAPGKERESVAHINRSGSSTYYAD9GS-23B04/155SVKGRFTISRDNAKNTVYLQLNSLKPEDTAVYYCAAGRYYSSDGVPSASFNYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBII86H09-EVQLVESGGGLVQAGGSLRLSCTASGSAFKSYRMGWFRRTPGKEDEFVASISWTYGSTFYAD9GS-23B04/156SVKGRFTMSRDKAKNAGYLQMNSLKPEDTALYYCAAGAQSDRYNIRSYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWG QGTQVTVSSVEGFBII87B07-EVQLVESGGGLVQPGGSLKLSCTASGFTFSTSWMHWVRQAPGKGLEWVSSIPPVGHFANYAP9GS-23B04/157SVKGRFTISRDNAKNTLFLQMNSLKSEDTAVYYCAKDSAGRTKGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVS SVEGFBII88A01-EVQLVESGGGLVQAGGSLRLSCAASERTFSNYAMDWFRQAPGKEREFVAAITRSGGGTYYAD9GS-23B04/158SVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAATRSSTIVVGVGGMEYWGKGTQVTVSSGGGGSGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBII4B08-EVQLVESGGGLVQPGGSLRLSCAASGSAVGDITVAWYRQAPGIQRQLVATITPSGYTYYWDF40GS-23B04/159VKGRFTISRDNSKNIVYLQMNSLKPEDTAAYYCNTQFYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBII5B03-EVQLVESGGGLAQAGDSLRLSCAASGRSFSHYNMGWFRQAPGKEREFVASIRGGGGSTTYAN40GS-23B04/160SVKDRFTISRENAKNTVYLQMNSLKPEDTAVYYCAATAFYRGPYDYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBII5B05-EVQLVESGGGLVQPGGSLRLSCVASGIRFMSMAWYRQAPGKHRELVARISSGGTTAYVDSVK40GS-23B04/161GRFTISRDNSKNTVYLQMNSLKAEDTAVYYCNTFSSRPNPWGAGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRIFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBII6G02-EVQLVESGGGLVQPGGSLRLSCAASGNIFSNNAMAWYRQAPGKQRELVARISSGGGFTYYLD40GS-23B04/162SVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCNAAYRTYNYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBII10E07- VEGFBII025EVQLVESGGGLVQAGGSLRLSCAASGRTFSNYAMGWFRQAPGKERVLVADISSSGINTYVAD40GS-23B04/163AVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASAWWYSQMARDNYRYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBII12801-EVQLVESGGGLVQPGGSLRLACAASGFTLSSSWMYWVRQAPGKGLEWVSRISPGGLFTYYVD40GS-23B04/164SVKGRFSVSTDNANNTLYLQMNSLKPEDTALYSCAKGGAPNYTPRGRGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBII86C11-EVQLVESGGGLVQAGDSLRLSCTASGRTFNSYAMGWFRQAPGKERESVAHINRSGSSTYYAD40GS-23B04/165SVKGRFTISRDNAKNTVYLQLNSLKPEDTAVYYCAAGRYYSSDGVPSASFNYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDANKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBII86H09-EVQLVESGGGLVQAGGSLRLSCTASGSAFKSYRMGWFRRTPGKEDEFVASISWTYGSTFYAD40GS-23B04/166SVKGRFTMSRDKAKNAGYLQMNSLKPEDTALYYCAAGAQSDRYNIRSYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBII87B07-EVQLVESGGGLVQPGGSLKLSCTASGFTFSTSWMHWVRQAPGKGLEWVSSIPPVGHFANYAP40GS-23B04/167SVKGRFTISRDNAKNTLFLQMNSLKSEDTAVYYCAKDSAGRTKGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBII88A01-EVQLVESGGGLVQAGGSLRLSCAASERTFSNYAMDWFRQAPGKEREFVAAITRSGGGTYYAD40GS-23B04/168SVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAATRSSTIVVGVGGMEYWGKGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS

The panel of 40 bivalent VHHs is tested in the VEGFR2 and VEGFR1blocking AlphaScreen assay, as described in Example 5.3 and 5.4,respectively. Based on potency and maximum level of inhibition, the 5best bivalent VHHs (VEGFBII021, VEGFBII022, VEGFBIO023, VEGFBIO24 andVEGFBII025) are chosen for further characterization. An overview of thescreening results for the 5 selected bivalent VHHs in the competitiveVEGFR2 and VEGFR1 AlphaScreen is shown in Table 16.

TABLE 16 Potency and efficacy of 5 best bivalent VHHs in the VEGF/VEGFR1and VEGF/VEGFR2 competition AlphaScreen assay VEGFR2 VEGFR1 VHH ID IC₅₀(pM) IC₅₀ (pM) % inhibition VEGFBII021 9 16 100 VEGFBII022 7 8 100VEGFBII023 38 44 91 VEGFBII024 12 46 100 VEGFBII025 51 39 82

Example 7 Characterization of Formatted Anti-VEGF VHHs

VHHs VEGFBII010, VEGFBII021, VEGFBII022, VEGFBII023, VEGFBII024 andVEGFBII025 are compared side-by-side in the VEGFR2 and VEGFR1 blockingELISA (FIGS. 8 and 9, Table 17 and Table 18 respectively) andAlphaScreen assay (FIGS. 10 and 11, Table 19 and 20) as described inExamples 5.1, 5.2, 5.3 and 5.4, respectively.

TABLE 17 IC₅₀ (pM) values and % inhibition for formatted VHHs inhVEGF165/hVEGFR2-Fc competition ELISA IC₅₀ VHH ID (pM) % inhibitionVEGFBII010 49 100 VEGFBII021 204 100 VEGFBII022 164 100 VEGFBII023 213100 VEGFBII024 292 100 VEGFBII025 577 100 Bevacizumab 315 100Ranibizumab 349 100

TABLE 18 IC₅₀ (pM) values and % inhibition of formatted VHHs inVEGF165/hVEGFR1-Fc competition ELISA IC₅₀ VHH ID (pM) % inhibitionVEGFBII010 73.5 67 VEGFBII021 254 97 VEGFBII022 225 89 VEGFBII023 279 91VEGFBII024 326 92 VEGFBII025 735 91 Bevacizumab 484 91 Ranibizumab 59496

TABLE 19 IC₅₀ (pM) values and % inhibition for formatted VHHs inhVEGF165/hVEGFR2-Fc competition AlphaScreen IC₅₀ VHH ID (pM) %inhibition VEGFBII010 16 100 VEGFBII021 7 100 VEGFBII022 7 100VEGFBII023 46 100 VEGFBII024 50 100 VEGFBII025 51 100 Ranibizumab 600100

TABLE 20 IC₅₀ (pM) values and % inhibition of formatted VHHs inVEGF165/hVEGFR1-Fc competition AlphaScreen IC₅₀ VHH ID (pM) % inhibitionVEGFBII010 21 70 VEGFBII021 12 100 VEGFBII022 9 98 VEGFBII023 48 87VEGFBII024 69 98 VEGFBII025 71 82 Ranibizumab 1300 87

In addition, formatted VHHs are also tested for their capacity to blockthe mVEGF164/mVEGFR2-huFc interaction. In brief, serial dilutions ofpurified VHHs (concentration range: 4 μM-14.5 pM) in PBS buffercontaining 0.03% Tween 20 (Sigma) are added to 0.1 nM biotinylatedmVEGF164 and incubated for 15 min. Subsequently mouse VEGFR2-huFc (0.1nM) and anti-huFc VHH-coated acceptor beads (20 μg/ml) are added andthis mixture is incubated for 1 hour. Finally, streptavidin donor beads(20 μg/ml) are added and after 1 hour of incubation fluorescence ismeasured on the Envision microplate reader. Dose-response curves areshown in FIG. 12. The IC₅₀ values for VHHs blocking the mouseVEGF164/VEGFR2-hFC interaction are summarized in Table 21.

TABLE 21 IC₅₀ (pM) values and % inhibition for formatted VHHs inmVEGF164/mVEGFR2-hFc competition AlphaScreen IC₅₀ VHH ID (nM) %inhibition VEGFBII022 108 100 VEGFBII024 — — mVEGF164 0.05 100Ranibizumab — —

The formatted VHHs are also tested in ELISA for their ability to bindmVEGF164 and human VEGF165 (Example 5.6; FIG. 13; Table 22); VEGF121(Example 5.7; FIG. 15; Table 23) and the VEGF family members VEGFB,VEGFC, VEGFD and PIGF (Example 5.8; FIG. 14). Binding kinetics for humanVEGF165 are analyzed as described in Example 5.5. The K_(D) values arelisted in Table 24.

TABLE 22 EC₅₀ (pM) values for formatted VHHs in a recombinant humanVEGF165 and mouse VEGF164 binding ELISA rhVEGF165 rmVEGF164 VHH ID EC₅₀(pM) EC₅₀ (pM) VEGFBII010 428 — VEGFBII021 334 502 VEGFBII022 224 464VEGFBII023 221 — VEGFBII024 320 — VEGFBII025 668 —

TABLE 23 EC₅₀ (pM) values for formatted VHHs in a recombinant humanVEGF121 binding ELISA rhVEGF121 VHH ID EC₅₀ (pM) VEGFBII010 920VEGFBII022 540 VEGFBII024 325 VEGFBII025 475

TABLE 24 Affinity K_(D) (nM) of purified formatted VHHs for recombinanthuman VEGF165 k_(a1) K_(D) VHH ID (1/Ms) k_(d1) (1/s) k_(a2) (1/s)k_(d2) (1/s) (nM)^((a)) VEGFBII010^((b)) 4.5E+05 1.7E−02 2.9E−02 1.3E−040.16 VEGFBII021^((b)) 1.2E+06 1.1E−02 2.3E−02 1.9E−04 0.07VEGFBII022^((b)) 1.2E+06 9.1E−03 1.4E−02 2.6E−04 0.14 VEGFBII023^((b))3.0E+05 1.8E−02 2.4E−02 2.7E−04 0.69 VEGFBII024^((b)) 3.0E+05 1.3E−022.6E−02 2.8E−04 0.47 VEGFBII025^((b)) 3.3E+05 1.7E−02 1.8E−02 3.7E−041.1 ^((a))K_(D) = k_(d1)/k_(a1) * (k_(d2)/(k_(d2) + k_(a2)))^((b))Curves are fitted using a Two State Reaction model by Biacore T100Evaluation Software v2.0.1

VHHs VEGFBII010, VEGFBII022, VEGFBII024 and VEGFBII025 are also testedin the VEGF-mediated HUVEC proliferation and Erk phosphorylation assay.

The potency of the selected formatted VHHs is evaluated in aproliferation assay. In brief, primary HUVEC cells (Technoclone) aresupplement-starved over night and then 4000 cells/well are seeded inquadruplicate in 96-well tissue culture plates. Cells are stimulated inthe absence or presence of VHHs with 33 ng/mL VEGF. The proliferationrates are measured by [³H]Thymidine incorporation on day 4. The resultsshown in Table 25 demonstrate that the formatted VHHs and Bevacizumabinhibit the VEGF-induced HUVEC proliferation by more than 90%, withIC₅₀s<1 nM.

TABLE 25 IC₅₀ (nM) values and % inhibition of formatted VHHs in VEGFHUVEC proliferation assay VHH ID IC₅₀ (nM) % inhibition VEGFBII010 0.2295 VEGFBII021 0.40 98 VEGFBII022 0.34 100 VEGFBII023 0.52 98 VEGFBII0240.38 96 VEGFBII025 0.41 104 Bevacizumab 0.21 92

The potency of the selected formatted VHHs is assessed in the HUVEC Erkphosphorylation assay. In brief, primary HUVE cells are serum-starvedover night and then stimulated in the absence or presence of VHHs with10 ng/mL VEGF for 5 min. Cells are fixed with 4% Formaldehyde in PBS andERK phosphorylation levels are measured by ELISA usingphosphoERK-specific antibodies (anti-phosphoMAP Kinase pERK1&2, M8159,Sigma) and polyclonal Rabbit Anti-Mouse-Immunoglobulin-HRP conjugate(PO161, Dako). As shown in Table 26, the formatted VHHs and Bevacizumabinhibit the VEGF induced Erk phosphoryaltion by more than 90%, withIC₅₀s<1 nM.

TABLE 26 IC₅₀ (nM) values and % inhibition of formatted VHHs in VEGFHUVEC Erk phosphorylation assay VHH ID IC₅₀ (nM) % inhibition VEGFBII0100.19 92 VEGFBII021 0.21 103 VEGFBII022 0.18 94 VEGFBII023 0.25 100VEGFBII024 0.23 94 VEGFBII025 0.23 99 Bevacizumab 0.63 98

Example 8 Sequence Optimization 8.1 Sequence Optimization ofVEGFBII23B04

The amino acid sequence of VEGFBII23B04 is aligned to the human germlinesequence VH3-23/JH5, see FIG. 16 (SEQ ID NO: 179)

The alignment shows that VEGFBII23B04 contains 19 framework mutationsrelative to the reference germline sequence. Non-human residues atpositions 14, 16, 23, 24, 41, 71, 82, 83 and 108 are selected forsubstitution with their human germline counterparts. A set of 8VEGFBII23B04 variants is generated carrying different combinations ofhuman residues at these positions (AA sequences are listed in Table 27).One additional variant is constructed in which the potentialisomerization site at position D59S60 (CDR2 region, see FIG. 16,indicated as bold italic residues) is removed by introduction of a S60Amutation.

TABLE 27  AA sequence of sequence-optimized variants of VHH VEGFBII23B04(FR, framework; CDR, complementary determining region) VHH ID/SEQ ID NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 VEGFBII111D05/ EVQLVESGG SYSMWFRQAPGKEREF AISKGGY RFTISRDNAKNTVYLQI SRAYGSS WGQGTLVTV 47 GLVQTGGSLR GVV KYDSVSL NSLRPEDTAVYYCAS RLRLADT SS LSCEASGRTF EG YEY S VEGFBII111G06/EVQLVESGG SYSM WFRQAPGKEREF AISKGGY RFTISRDNAKNTVYLQM SRAYGSS WGQGTLVTV48 GLVQPGGSL G VV KYDSVSL NSLRPEDTAVYYCAS RLRLADT SS RLSCAASGRT EG YEYFS VEGFBII112D11/ EVQLVESGG SYSM WFRQAPGKEREF AISKGGY RFTISRDNAKNTVYLQISRAYGSS WGQGTLVTV 49 GLVQPGGSL G VV KYDSVSL NSLRPEDTAVYYCAS RLRLADT SSRLSCEASGRT EG YEY FS VEGFBII113A08/ EVQLVESGG SYSM WFRQAPGKEREF AISKGGYRFTISKDNAKNTVYLQIN SRAYGSS WGQGTLVTV 50 GLVQTGGSLR G VV KYDSVSLSLRPEDTAVYYCAS RLRLADT SS LSCEVSGRTF EG YEY S VEGFBII113E03/ EVQLVESGGSYSM WFRQAQGKERE AISKGGY RFTISKDNAKNTVYLQM SRAYGSS WGQGTLVTV 51GLVQTGDSLR G FVV KYDSVSL NSLRPEDTAVYYCAS RLRLADT SS LSCEVSGRTF EG YEY SVEGFBII114C09/ EVQLVESGG SYSM WFRQAPGKEREF AISKGGY RFTISKDNAKNTVYLQINSRAYGSS WGQGTLVTV 52 GLVQPGDSLR G VV KYDSVSL SLRPEDTAVYYCAS RLRLADT SSLSCEVSGRTF EG YEY S VEGFBII114D02/ EVQLVESGG SYSM WFRQAPGKEREF AISKGGYRFTISRDNAKNTVYLQI SRAYGSS WGQGTLVTV 53 GLVQTGGSLR G VV KYDSVSLNSLRPEDTAVYYCAS RLRLADT SS LSCEVSGRTF EG YEY S VEGFBII114D03/ EVQLVESGGSYSM WFRQAQGKERE AISKGGY RFTISKDNAKNTVYLQIN SRAYGSS WGQGTLVTV 54GLVQTGDSLR G FVV KYDSVSL SLRPEDTAVYYCAS RLRLADT SS LSCAVSGRTF EG YEY SVEGFBII118E10/ EVQLVESGG SYSM WFRQAQGKERE AISKGGY RFTISKDNAKNTVYLQINSRAYGSS WGQGTQVTV 55 GLVQTGDSLR G FVV KYDAVSL SLKPEDTAVYYCAS RLRLADT SSLSCEVSGRTF EG YEY S

These variants are characterized as purified proteins in theVEGF165/VEGFR2 AlphaScreen (Example 5.3, FIG. 17). The meltingtemperature (T_(m)) of each clone is determined in a thermal shiftassay, which is based on the increase in fluorescence signal uponincorporation of Sypro Orange (Invitrogen) (Ericsson et al, Anal.Biochem. 357 (2006), pp 289-298). All variants displayed comparable IC₅₀when compared to VEGFBII23B04 and T_(m) values which are similar orhigher when compared to the parental VEGFBII23B04. Table 28 summarizesthe IC₅₀ values and T_(m) values at pH 7 for the 9 clones tested.

TABLE 28 IC₅₀ (pM) values, % inhibition and melting temperature (@ pH 7)of sequence-optimized variants of VEGFBII23B04 VHH ID IC₅₀ (pM) %inhibition T_(m) @ pH 7 (° C.) VEGFBII23B04 (wt) 169 100 63VEGFBII111D05 209 100 68 VEGFBII111G06 366 100 71 VEGFBII112D11 221 10070 VEGFBII113A08 253 100 69 VEGFBII113E03 290 100 68 VEGFBII114C09 215100 71 VEGFBII114D02 199 100 74 VEGFBII114D03 227 100 64 VEGFBII118E10189 100 62

In a second cycle, tolerated mutations from the humanization effort(VEGFBII111G06) and mutations to avoid potential posttranslationalmodification at selected sites (the D16G, the S60A substitution and anE1D mutation) are combined resulting in a sequence-optimized clonederived from VEGFBII23B04: VEGFBII0037. One extra sequence-optimizedvariant (VEGFBII038) is anticipated which contains the samesubstitutions as VEGFBII0037, with the exception of the 182M mutation,as this mutation may be associated with a minor drop in potency. Thesequences from both sequence-optimized clones are listed in Table 29.VEGFBII0037 and VEGFBII0038 are characterized in the VEGF165/VEGFR2blocking AlphaScreen (Example 5.3, FIG. 18), the melting temperature isdetermined in the thermal shift assay as described above and theaffinity for binding on VEGF165 is determined in Biacore (Example 5.5).An overview of the characteristics of the 2 sequence-optimized VHHs ispresented in Table 30.

TABLE 29 AA sequences of sequence-optimized variants of VHH VEGFBII23B04 VHH ID/SEQ ID CDR NO: FR 1 1 FR2 CDR 2 FR3 CDR 3 FR 4 VEGFBII0 DVQLVES SYSMWFRQA AISKGG RFTISRDNAKNT SRAYGSSR  WGQGT 37 GGGLVQP G PGKERE YKYDAVYLQMNSLRPE LRLADTYEY LVTVSS 56 GGSLRLS FVV VSLEG DTAVYYCAS CAASGRT FSVEGFBII0 DVQLVES SYSM WFRQA AISKGG RFTISRDNAKNT SRAYGSSR WGQGT 38GGGLVQP G PGKERE YKYDA VYLQINSLRPED LRLADTYEY LVTVSS 57 GGSLRLS FVVVSLEG TAVYYCAS CAASGRT FS

TABLE 30 IC₅₀ (pM) values, % inhibition, melting temperature (@pH 7) andaffinity (pM) of sequence-optimized clones VEGFBII037 and VEGFBII038 %T_(m) (° C.) VHH ID IC₅₀ (pM) inhibition @ pH 7 K_(D) (pM) VEGFBII23B04152 100 63 560 VEGFBII037 300 100 72 270 VEGFBII038 143 100 71 360

8.2 Sequence Optimization of VEGFBII5B05

The amino acid sequence of VEGFBII5B05 is aligned to the human germlinesequence VH3-23/JH5, see FIG. 19 (SEQ ID:NO: 179 The alignment showsthat VEGFBII5B05 contains 15 framework mutations relative to thereference germline sequence. Non-human residues at positions 23, 60, 83,105, 108 are selected for substitution with their human germlinecounterparts while the histidine at position 44 is selected forsubstitution by glutamine. One humanization variant is constructedcarrying the 6 described mutations (AA sequence is listed in Table 31).

TABLE 31  AA sequences of sequence-optimized variants of VHH VEGFBII5B05(FR, framework; CDR, complementary determining region) VHH ID/SED ID NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 VEGFBII119G11/ EVQLVES SMAWYRQAP RISSG RFTISRDNSK FSSRP WGQGT 125 GGGLVQP GKQRELV GTTA NTVYLQMNSNP LVTVSS GGSLRLS A YADS LRAEDTAVY CAASGIR VKG YCNT FM VEGFBII120E10/EVQLVES SMA WYRQAP RISSG RFTISRDNSK FSSRP WGAGT 126 GGGLVQP GKHRELV GTTANTVYLQMNS NP QVTVS GGSLRLS A YVDS LKAEDTAVY S CVASGIR VKG YCNT FI

One additional variant is constructed in which the potential oxidationsite at position M30 (CDR1 region, see FIG. 19 indicated as bold italicresidue) is removed by introduction of a M301 mutation. Both variantsare tested for their ability to bind hVEGF165 using the ProteOn. Inbrief, a GLC ProteOn Sensor chip is coated with human VEGF165.Periplasmic extracts of the variants are diluted 1/10 and injectedacross the chip coated with human VEGF165. Off-rates are calculated andcompared to the off-rates of the parental VEGFBII5B05. Off-rates fromthe 2 variants are in the same range as the off-rates from the parentalVEGFBII5B05 indicating that all mutations are tolerated (Table 32).

TABLE 32 Off-rates sequence-optimized variants VEGFBII5B05 VHH IDbinding level (RU) k_(d) (1/s) VEGFBII5B05 242 6.15E−02 VEGFBII119G11234 7.75E−02 VEGFBII120E10 257 4.68E−02

In a second cycle, mutations from the humanization effort and the M301substitution are combined resulting in a sequence-optimized clone ofVEGFBII5B05, designated VEGFBII032. The sequence is listed in Table 33.Affinity of VEGFBII032 is determined by Biacore (see Example 5.5) andthe melting temperature is determined in the thermal shift assay asdescribed above. An overview of the characteristics of thesequence-optimized VHH VEGFBII032 is presented in Table 34.

TABLE 33  AA sequence of sequence-optimized clone VEGFBII032(FR, framework; CDR, complementary determining region) VHH ID/SEQ ID NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 VEGFBII032/ EVQLVES SMA WYRQAPRISSG RFTISRDNSK FSSR WGQGTL 127 GGGLVQP GKQRELV GTTA NTVYLQMNS PNPVTVSS GGSLRLS A YADS LRAEDTAVY CAASGIR VKG YCNT FI

TABLE 34 Melting temperature (@pH 7) and affinity (nM) ofsequence-optimized clone VEGFBII032 T_(m) (° C.) VHH ID @ pH 7 K_(D)(nM) VEGFBII5B05(wt) 69 32 VEGFBII0032 71 44

The potency of the sequence-optimized clones VEGFBII037 and VEGFBII038is evaluated in a proliferation assay. In brief, primary HUVEC cells(Technoclone) are supplement-starved over night and then 4000 cells/wellare seeded in quadruplicate in 96-well tissue culture plates. Cells arestimulated in the absence or presence of VHHs with 33 ng/mL VEGF. Theproliferation rates are measured by [³H]Thymidine incorporation on day4. The results shown in Table 35, demonstrate that the activity (potencyand degree of inhibition) of the parental VHH VEGFBII23B04 is conservedin the sequence optimized clone VEGFBII038.

TABLE 35 IC₅₀ (nM) values and % inhibition of the sequence optimizedclones VEGFBII037 and VEGFBII038 in VEGF HUVEC proliferation assay VHHID IC₅₀ (nM) % inhibition VEGFBII23B04 0.68 92 VEGFBII037 1.54 78VEGFBII038 0.60 92 Bevacizumab 0.29 94

Example 9 Construction, Production and Characterization of Bivalent VHHsTargeting Ang2

VHHs 1D01 (SEQ ID No:214), 11B07, 00908 and 00027 (SEQ ID No:216) aregenetically fused to 1 D01 (SEQ ID No: 214), 11807, 00908 and 00027 (SEQID No:216), respectively, resulting in homodimeric VHHs. The bivalentVHHs are linked via a 9-GlySer or 40-GlySer flexible linker. Theencoding DNA sequences of the formatted VHHs are cloned in theexpression vector pAX172. VHHs are expressed in Pichia pastoris asc-terminally myc-His6 tagged proteins. In brief, BGCM cultures arestarted from a single colony streak incubated over weekend at 30° C.(250 rpm). After medium switch to BMCM, cultures are incubated untilevening at 30° C. (250 rpm) and followed by an induction with 100%methanol. The next day the cultures are induced an additional 3 times(morning, afternoon, evening). The next day cultures are centrifuged for20 min at 4° C. (1,500×g). The His6-tagged VHHs present in thesupernatant are purified through immobilized metal affinitychromatography (IMAC) followed by desalting (DS) and finally gelfiltration (GF) to remove any endotoxins/impurities. An overview of theformat and sequence of all bivalent VHHs is depicted in FIG. 20 andTable 36-A (linker sequences underlined), SEQ ID Nos 180-185. Expressionlevels are indicated in Table 36-B.

To explore the anti-Ang2 blocking properties in comparison with themonovalent building blocks, bivalent VHHs are analyzed in a humanAng2/hTie2 (FIG. 21-1), mouse Ang2/mTie2 (FIG. 21-2), cyno Ang2/cTie2(FIG. 21-3) and human Ang1/hTie2 (FIG. 22) competition ELISA. A summaryof IC₅₀ values is shown in Table 37.

TABLE 36-A  Sequences of bivalent VHH targeting Ang2 VHH ID AA sequenceANGBII00001EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSS (SEQ IDNO: 180) ANGBII00002EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYENDAWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSS (SEQ ID NO: 181) ANGBII00003EVQLVESGGGLVQVGDSLRLSCAASGRTFSTYLMVGWFRQAPGKEREFAAGIWSSGDTAYADSVRGRFTISRDNAKNTVYLQMNSLKTEDTAVYYCAGSYDGNYYIPGFYKDWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQVGDSLRLSCAASGRTFSTYLMVGWFRQAPGKEREFAAGIWSSGDTAYADSVRGRFTISRDNAKNTVYLQMNSLKTEDTAVYYCAGSYDGNYYIPGYYKDWGQGTLVTVSS (SEQ ID NO: 182)ANGBII00004EVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS(SEQ ID NO: 183) ANGBII00005EVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWITLYEYDAWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS (SEQ ID NO: 184) ANGBII00006EVQLVESGGGLVQPGGSLRLSCAASGITLDDYAIGWFRQAPGKEREGVSSIRDNGGSTYYADSVKGRFTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSSGGGGSGGGSEVQLLESGGGLVQPGGSLRLSCAASGITLDDYAIGWFRQAPGKEREGVSSIRDNGGSTYYADSVKGRFTISSQNSENTVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS(SEQ ID NO: 185)

Example 10 Construction, Production and Characterization of TrivalentBispecific VHHs Targeting VEGF and Ang2 Using Anti-Serum Abumin asHalf-Life Extension

The anti-VEGF VHH VEGFBII00038 (US 2011/0172398 A1) and the anti-Ang2VHH 00027 (SEQ ID No:216) are used as building blocks to generatebispecific VHHs VEGFANGBII00001-00004. A genetic fusion to a serumalbumin binding VHH is used as half-life extension methodology. Buildingblocks are linked via a triple Ala or 9 Gly-Ser flexible linker. VHHsare produced and purified as described in Example 9. An overview of theformat and sequence of all four bispecific VHHs is depicted in FIG. 23and Table 37-A (linker sequences underlined), SEQ ID Nos 186-189.Expression levels are indicated in Table 38-B.

TABLE 38-A  Sequences of bispecific VHH targeting VEGF and Ang2 VHH IDAA sequence VEGFANGBII00001DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS (SEQ ID NO: 186)VEGFANGBII00002EVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSDVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSS (SEQ ID NO: 187)VEGFANGBII00003EVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGSGGGSDVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 188)VEGFANGBII00004DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSAAAEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSAAAEVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS (SEQ ID NO: 189)

To explore the anti-VEGF blocking properties in comparison with themonovalent building block VEGFBII00038, all four bispecific VHHs areanalyzed in the VEGF/VEGFR2-Fc (FIG. 22) competition AlphaScreen. Theassay is slightly adjusted compared to Example 12.3 described in patentUS 2011/0172398 A1. Both human VEGF165 and human VEGFR2-Fc are added at0.05 nM. This competition assay is also performed after preincubation ofthe VHH with 25 μM human serum albumin. A summary of IC₅₀ values and %inhibition is shown in Table 39.

To explore the anti-Ang2 blocking properties in comparison with themonovalent building block 00027 (SEQ ID No:216), all four bispecificVHHs are analyzed in a human Ang2/hTie2-Fc (FIG. 25) competition ELISA.This assay is also performed after incubation of the VHH with 0.5 μMhuman serum albumin. A summary of IC₅₀ values is shown in Table 40.

Example 11 Construction, Production and Characterization of Trivalentand Tetravalent Bispecific VHHs Targeting VEGF and Ang2 Using Anti-SerumAlbumin Binding as Half-Life Extension

Ten bispecific VHHs targeting VEGF and Ang2 are constructed(VEGFANGBII00005-00015). In these constructs monovalent and bivalent1D01 (SEQ ID NO:214), monovalent and bivalent 7G08 (SEQ ID NO:215) andbivalent 00027 (SEQ ID NO:216) anti-Ang2 building blocks are included. Agenetic fusion to a serum albumin binding VHH is used as half-lifeextension methodology. Building blocks are linked via a 9 Gly-Serflexible linker. VHHs are produced and purified as described in Example8. An overview of the format and sequence of all ten bispecific VHHs isdepicted in FIG. 26 and Table 41-A (linker sequences underlined), SEQ IDNos 190-199. Expression levels are indicated in Table 41-B.

TABLE 41-A  Sequences of bispecific VHH targeting VEGF and Ang2 VHH IDAA sequence VEGFANGBII00005DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFALDYYAIGWFRQVPGKEREGVSCISSSDGITYYVDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDSGGYIDYDCMGLGYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 190)VEGFANGBII00006DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFALDYYAIGWFRQVPGKEREGVSCISSSDGITYYVDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDSGGYIDYDCMGLGYDYWGQGTLVTVSS (SEQ ID NO: 191)VEGFANGBII00007DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFALDYYAIGWFRQVPGKEREGVSCISSSDGITYYVDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDSGGYIDYDCMGLGYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFALDYYAIGWFRQVPGKEREGVSCISSSDGITYYVDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDSGGYIDYDCMGLGYDYWGQGTLVTVSS (SEQ ID NO: 192)VEGFANGBII00008DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLAPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ IDNO: 193) VEGFANGBII00009DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS (SEQ IDNO: 194) VEGFANGBII00010DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS (SEQ IDNO: 195) VEGFANGBII00011DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 196)VEGFANGBII00012DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTTYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSS (SEQ ID NO: 197)VEGFANGBII00013DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSS (SEQ ID NO: 198)VEGFANGBII00014DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSS (SEQ ID NO: 199)

To explore the anti-VEGF blocking properties in comparison with themonovalent building block VEGFBII00038, all ten bispecific VHHs areanalyzed in the VEGF/VEGFR2-Fc (Example 10; FIG. 27-1), and VEGF/VEGFR1(FIG. 27-2) competition AlphaScreen. The VEGFR1 assay is slightlyadjusted compared to Example 12.4 as described in patent US 2011/0172398A1. Human VEGF165 and human VEGFR1-Fc are added at 0.05 nM. Thesecompetition assays are also performed after preincubation of the VHHwith 25 μM human serum albumin. A summary of IC₅₀ values is shown inTable 42.

To explore the anti-Ang2 blocking properties in comparison with theirrespective monovalent building block 7G08 (SEQ ID No:215), 1D01 (SEQ IDNo:214) and 00027 (SEQ ID No:216), all ten bispecific VHHs are analyzedin the human Ang2/hTie2-Fc (see Example 5.1; FIG. 28-1), mouseAng2/mTie2-Fc (see Example 5.2; FIG. 28-2) and cyno Ang2/cTie2-Fc (seeExample 5.2; FIG. 28-3) competition ELISA. The human assay is alsoperformed after incubation of the VHH with 0.5 μM human serum albumin.Additionally, a hAng2 mediated HUVEC survival assay is performed (seeExample 5.5; FIG. 29). A summary of IC₅₀ values and % inhibition isshown in Table 43.

Affinities of for human serum albumin have been determined and are shownin Table 44. Briefly, human serum albumin (Sigma, St Louis, Mo., USA) isimmobilized on a CM5 chip via amine coupling. A multicycle kineticapproach is used: increasing concentrations of VHH (2-8-31-125-500 nM)are injected and allowed to associate for 2 min and to dissociate for 10min at a flow rate of 100 μL/min. Between VHH injections, the surfacesare regenerated with a 10 sec pulse of 10 mM Glycine-HCl pH 1.5 and 60sec stabilization period. Association/dissociation data are evaluated byfitting a 1:1 interaction model (Langmuir binding) or HeterogeneousLigand model. The affinity constant K_(D) is calculated from resultingassociation and dissociation rate constants k_(a) and k_(d) (Table 44).

TABLE 44 Affinity K_(D) of purified VHHs for human (HSA), cyno (CSA) andmouse serum albumin (MSA) HSA CSA MSA k_(a) k_(d) K_(D) k_(a) k_(d)K_(D) k_(a) k_(d) K_(D) (1/Ms) (1/s) (nM) (1/Ms) (1/s) (nM) (1/Ms) (1/s)(nM) ALB11 4.5E+05 1.8E−03  4 4.3E+05 1.6E−03  4 6.6E+05 3.2E−02  49VEGFANGBII00001 2.3E+05 4.8E−03 22 1.8E+05 4.3E−03 24 n.d. n.d. n.d.VEGFANGBII00005 n.d. n.d. n.d. n.d n.d n.d. n.d. n.d. n.d.VEGFANGBII00006 2.0E+05 4.6E−03 22 1.5E+05 4.5E−03 30 1.7E+05 6.0E−02360 VEGFANGBII00007 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.VEGFANGBII00008 1.3E+05 4.3E−03 34 n.d. n.d. n.d. n.d. n.d. n.d.VEGFANGBII00009 1.5E+05 4.6E−03 30 1.1E+05 4.2E−03 39 1.2E+05 4.0E−02340 VEGFANGBII00010 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.VEGFANGBII00011 1.3E+05 4.0E−03 31 n.d. n.d. n.d. n.d. n.d. n.d.VEGFANGBII00012 1.5E+05 4.3E−03 31 1.2E+05 4.2E−03 24 1.0E+05 2.5E−02240 VEGFANGBII0013 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.VEGFANGBII0014 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d., notdetermined

Example 12 Construction, Production and Characterization of SequenceOptimized and Affinity Matured Bispecific VHHs Targeting VEGF and Ang2Using Anti-Serum Albumin Binding as Half-Life Extension

14 bispecific VHHs targeting VEGF and Ang2 are constructed(VEGFANGBII00015-00028). In these constructs bivalent 00921 (a sequenceoptimized 1D01 variant) (SEQ ID No:220), monovalent VHHs00908-00932-00933-00934-00935-00936-00937-00938 (sequenceoptimized/affinity matured 28D10 variants) (SEQ ID No:222), bivalent00956 (SEQ ID NO:223) (sequence optimized 28D10 variant) and monovalent00928 (SEQ ID NO:221) (sequence optimized 37F02 variant) anti-Ang2building blocks are included. A genetic fusion to a serum albuminbinding VHH is used as half-life extension methodology. Building blocksare linked via a 9 Gly-Ser flexible linker. An overview of the formatand sequence of all 14 bispecific VHHs is depicted in FIG. 30 and Table45-A (linker sequences underlined), SEQ ID Nos 200-213. Expressionlevels are indicated in Table 45-B.

TABLE 45-A Sequences of bispecific VHH targeting VEGF and Ang2 VHH IDAA sequence VEGFANGBII00015DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGITLDDYAIGWFRQAPGKEREGVSSIRDNGGSTYYADSVKGRFTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS (SEQ ID NO: 200)VEGFANGBII00016DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAVSGITLDDYAIGWFRQAPGKEREGVSSIRDNGGSTYYADSVKGRFTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: 201)VEGFANGBII00017DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGITLDDYAIGWFRQAPGKEREGVSAIRDNGGSTYYADSVKGRFTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: 202)VEGFANGBII00018DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGITLDDYAIGWFRQAPGKEREGVSAIRESGGSTYYADSVKGRFTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: 203)VEGFANGBII00019DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGRGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGITLDDYAIGWFRQAPGKEREGVSAIRSSGGSTYYADSVKGRFTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: 204)VEGFANGBII00020DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAVSGITLDDYAIGWFRQAPGKEREGVSAIRDNGGSTYYADSVKGRFTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: 205)VEGFANGBII00021DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAVSGITLDDYAIGWFRQAPGKEREGVSAIRESGGSTYYADSVKGRFTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: 206)VEGFANGBII00022DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGRGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAVSGITLDDYAIGWFRQAPGKEREGVSAIRSSGGSTYYADSVKGRFTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: 207)VEGFANGBII00023DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGREREFVVAISKGGYKYDAVSLEGRFTISRDNARNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSAIRDNGGSTYYADSVKGRFTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSAIRDNGGSTYYADSVKGRFTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS SEQ ID NO: 208)VEGFANGBII00024DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSAIRDNGGSTYYADSVKGRFTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGREREGVSAIRDNGGSTYYADSVKGRFTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS SEQ ID NO: 209)VEGFANGBII00025DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYALGWFRQAPGKEREGVSCIRCSGGSTYYADSVKGRFTISSDNSKNTVYLQMNSLRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYALGWFRQAPGKEREGVSCIRCSGGSTYYADSVKGRFTISSDNSKNTVYLQMNSLRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSS SEQ ID NO: 210)VEGFANGBII00026DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYALGWFRQAPGKEREGVSCIRCSGGSTYYADSVKGRFTISSDNSKNTVYLQMNSLRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYALGWFRQAPGKEREGVSCIRCSGGSTYYADSVKGRFTISSDNSKNTVYLQMNSLRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS SEQ ID NO: 211)VEGFANGBII00027DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFALDYYAIGWFRQAPGKEREGVSCISSSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCATDSGGYIDYDCSGLGYDYWGQGTLVTVSS SEQ ID NO: 212)VEGFANGBII00028DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSAIRSSGGSTYYADSVKGRFTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSAIRSSGGSTYYADSVKGRFTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS SEQ ID NO: 213)

To explore the anti-VEGF blocking properties in comparison with themonovalent building block VEGFBII00038, the bispecific VHHs are analyzedin the VEGF/VEGFR2-Fc (Example 10; FIG. 31-1) and VEGF/VEGFR1 (Example11; FIG. 31-2) competition AlphaScreen. These competition assays arealso performed after preincubation of the VHH with 25 μM human serumalbumin. A summary of IC₅₀ values is shown in Table 46-A.

Binding kinetics of the bispecific VHHs on human VEGF165 is analyzed bySPR on a Biacore T100 instrument (see Example 12.5 described in patentUS 2011/0172398 A1). Monovalent Nanobody VEGFBII00038 is taken along asreference (Table 46-B).

TABLE 46-B Overview of kinetic parameters in hVEGF165 Biacore assay. ka2kd2 KD1 ka1 (1/Ms) kd1 (1/s) (1/s) (1/s) (M) VEGFBII00038 2.6E+051.3E−02 1.3E−02 1.9E−04 7.5E−10 VEGFANGBII00022 1.6E+05 1.4E−02 1.4E−022.2E−04 1.4E−09 VEGFANGBII00025 1.1E+05 1.4E−02 1.4E−02 2.1E−04 1.9E−09VEGFANGBII00028 1.7E+05 1.3E−02 1.3E−02 2.1E−04 1.1E−09

The ability of the VHHs to bind to human isoform VEGF121 is determinedin a binding ELISA. Binding of a dilution series of VHH to 1 μg/mLdirectly coated human VEGF121 (R&D) (human VEGF165 as reference) isdetected using biotinylated anti-VHH 1A4 followed by extravidin-HRP. 1A4is a anti-VHH VHH (generated in-house by Ablynx NV). The benchmarkAvastin serves as positive control and is detected using a HRPconjugated anti-human Fc antibody. An Irrelevant VHH serves as negativecontrol. Representative binding response curves on VEGF165 and VEGF121are shown in FIG. 46 corresponding EC₅₀ values are summarized in Table46-C.

TABLE 46-C Overview of EC₅₀ values in hVEGF165 and hVEGF121 bindingELISA. hVEGF165 hVEGF121 EC₅₀ (M) EC₅₀ (M) VEGFANGBII00022 1.4E−092.3E−09 VEGFANGBII00025 1.5E−09 2.5E−09 VEGFANGBII00028 1.2E−09 2.1E−09

Binding to rat and mouse VEGF164 is assessed in a binding ELISA. VHHsbinding to 1 μg/mL directly coated murine or rat VEGF164 (R&D) aredetected using biotinylated anti-VHH 1A4 followed by extravidin-HRP. Aspositive control the mouse/rat cross-reactive monoclonal antibodyB20-4.1 (Genentech) is titrated and detected with an HRP conjugatedanti-human Fc antibody. An irrelevant VHH serves as negative control.Results are shown in FIG. 33. All 3 bispecific VHH are notcross-reactive to mouse and rat VEGF.

Binding to human VEGF-B, VEGF-C, VEGF-D and PIGF is assessed via abinding ELISA. Binding of VHHs to 1 μg/mL directly coated VEGF-B (R&D),VEGF-C(R&D), VEGF-D (R&D) and PIGF (R&D) was detected using biotinylatedanti-VHH 1A4 followed by extravidin-HRP. As positive controls a seriesof dilutions of the appropriate receptors (hVEGFR1-Fc for hVEGF-B andhPIGF, hVEGFR2-Fc for hVEGF-C, anti-hVEGF-D mAb (R&D) for hVEGF-D) aretaken along. An irrelevant VHH serves as negative control. Results areshown in FIG. 34. All 3 bispecific VHH are not binding to VEGF familymembers.

To explore the anti-Ang2 blocking properties in comparison with theirrespective monovalent building block 00921 (SEQ ID NO:220) and 00938(SEQ ID NO:222), all 3 bispecific VHHs are analyzed in the humanAng2/hTie2-Fc (see Example 5.1; FIG. 35-1), mouse Ang2/mTie2-Fc (seeExample 5.2; FIG. 35-2) and cyno Ang2/cTie2-Fc (see Example 5.2; FIG.35-3) competition ELISA. The human assay is also performed afterincubation of the VHH with 0.5 μM human serum albumin. Additionally,bispecific VHHs are tested in the hAng1/hTie2 competition ELISA (seeExample 5.3; FIG. 36) and the Ang2 mediated HUVEC survival assay (seeExample 5.5; FIG. 37). A summary of IC₅₀ values and % inhibition isshown in Table 47-A.

Affinities of VEGFANGBII00022-25-28 for human, mouse, cyno and rat Ang2(see Example 5.4) have been determined and are shown in Table 47-B.

TABLE 47-B Affinity KD of purified VHHs for recombinant human, cyno,mouse and rat Ang2 human Ang2-FLD cyno Ang2-FLD k_(a) k_(d) K_(D) k_(a)k_(d) K_(D) (1/Ms) (1/s) (M) (1/Ms) (1/s) (M) VEGFANGBII00022 9.7E+051.5E−05 1.6E−11 1.5E+06 1.3E−05 8.1E−12 VEGFANGBII00025 2.7E+06 1.2E−024.5E−09 4.3E+06 1.1E−02 2.7E−09 VEGFANGBII00028 5.9E+05 9.6E−04 1.6E−098.4E+05 8.7E−04 1.0E−09 mouse Ang2-FLD rat Ang2-FLD k_(a) k_(d) K_(D)k_(a) k_(d) K_(D) (1/Ms) (1/s) (M) (1/Ms) (1/s) (M) VEGFANGBII000225.5E+05 2.8E−05 5.1E−11 3.9E+05 3.8E−05 9.9E−11 VEGFANGBII00025 1.3E+061.4E−02 1.1E−08 8.7E+05 2.9E−02 3.3E−08 VEGFANGBII00028 3.6E+05 2.0E−035.6E−09 2.5E+05 3.1E−03 1.2E−08

Affinities of VEGFANGBII00022-25-28 for human, mouse and cyno serumalbumin have been determined (Example 11) and are shown in Table 48. Theaffinity constant K_(D) is calculated from resulting association anddissociation rate constants k_(a) and k_(d) (Table 48).

TABLE 48 Affinity KD (nM) of purified VHHs for recombinant human, mouseand cyno serum albumin using (A) 1:1 interaction model or (B)heterogeneous ligand model (A) HSA CSA k_(a) k_(d) K_(D) k_(a) k_(d)K_(D) (1/Ms) (1/s) (nM) (1/Ms) (1/s) (nM) ALB11 5.6E+05 1.9E−03 44.5E+05 1.7E−03 4 VEGFANGBII00022 6.7E+05 6.0E−03 9 6.2E+05 5.4E−03 9VEGFANGBII00025 5.6E+05 5.6E−03 12 4.3E+05 5.1E−03 12 VEGFANGBII000285.6E+05 5.8E−03 10 5.2E+05 5.3E−03 10 MSA k_(a) k_(d) K_(D) (1/Ms) (1/s)(nM) ALB11 5.9E+05 3.0E−02 51 VEGFANGBII00022 5.2E+05 5.4E−03 150VEGFANGBII00025 — — — VEGFANGBII00028 — — — (B) MSA k_(a1) k_(d1) k_(a2)k_(d2) K_(D1) K_(D2) (1/Ms) (1/s) (1/s) (1/s) (nM) (nM) VEGFANGBII000256.2E+05 9.9E−02 4.7E+04 5.7E−04 160 * 12  VEGFANGBII00028 5.9E+046.9E−04 5.7E+05 9.4E−02 12  160 * * describes 70% or more of theinteraction

TABLE 49 Ang2-binding components(1D01 (SEQ ID No: 214); 7G08 (SEQ ID No: 215); 027 (SEQ ID No: 216); 00042(SEQ ID No: 217); 00050 (SEQ ID No: 218); 00045 (SEQ ID No: 219); 00921(SEQ ID No: 220); 00928 (SEQ ID No: 221); 00938 (SEQ ID No: 222); 00956 (SEQ ID No: 223)FR1 CDR1 FR2 CDR2 1D01 EVQLVESGGGLVQAGGSLRLSCAASGFTFD DYALDWFRQAAGKEREGVS CIRCSDGSTYYADSVKG 7G08 EVQLVESGGGLVQPGGSLRLSCAASGFALDYYAIG WFRQVPGKEREGVS CISSSDGITYYVDSVKG   027EVQLVESGGGLVQAGGSLRLSCAASGFTLD DYAIG WFRQAPGKEREGVS CIRDSDGSTYYADSVKGFR3 CDR3 FR4 1D01 RFTISSDNAKNTVYLQMNSLKPEDTAVYYCAA SIVPRSKLEPYEYDAWGQGTQVTVSS 7G08 RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAT DSGGYIDYDCMGLGYDYWGQGTQVTVSS   027 RFTISSDNAKNTVYLQMNSLKPEDTAVYYCAA VPAGRLRFGEQWYPLYEYDAWGQGTQVTVSS FR1 CDR1 FR2 CDR2 00042 EVQLVESGGGLVQAGGSLRLSCAASGFTFD DYALDWFRQAAGKEREGVS SIRDNDGSTYYADSVKG 00050 EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIG WFRQAPGKEREGVS CIRCSDGSTYYVDSVKG 00045EVQLVESGGGLVQAGGSLRLSCAASGFALD DYAIG WFRQAPGKEREGVS CISSSDGITYYADSVKGFR3 CDR3 FR4 00042 RFTISSDNSKNTVYLQMNSLKPEDTAVYYCAA VPAGRLRFGEQWYPLYEYDAWGQGTQVTVSS 00050 RFTISRDNSKNTVYLQMNSLKPEDTAVYYCAA SIVPRSKLEPYEYDAWGQGTQVTVSS 00045 RFTISRDNSKNTVYLQMNSLKPEDTAVYYCAT DSGGYIDYDCMGLGYDYWGQGTQVTVSS FR1 CDR1 FR2 CDR2 00921 EVQLVESGGGLVQPGGSLRLSCAASGFTFD DYALDWFRQAPGKEREGVS CIRCSGGSTYYADSVKG 00928 EVQLVESGGGLVQPGGSLRLSCAASGFALDYYAIG WFRQAPGKEREGVS CISSSGGITYYVDSVKG 00938EVQLVESGGGLVQPGGSLRLSCAVSGITLD DYAIG WFRQAPGKEREGVS AIRSSGGSTYYADSVKG00956 EVQLVESGGGLVQPGGSLRLSCAASGFTLD DYAIG WFRQAPGKEREGVSAIRSSGGSTYYADSVKG FR3 CDR3 FR4 00921 RFTISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDA WGQGTQVTVSS 00928 RFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDSGGYIDYDCSGLGYDY WGQGTQVTVSS 00938 RFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDA WGQGTQVTVSS 00956 RFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDA WGQGTQVTVSS

1. A bispecific binding molecule comprising at least one VEGF-bindingcomponent, at least one Ang2-binding component and at least one serumalbumin binding component, wherein said Ang2-binding component binds toAng2 with a potency at least 5,000 times higher than to Ang1 or to Ang4.2. A bispecific binding molecule of claim 1, wherein said VEGF-bindingcomponent comprises at least a variable domain with four frameworkregions and three complementarity determining regions CDR1, CDR2 andCDR3, respectively, wherein said CDR3 has the amino acid sequence SerArg Ala Tyr Xaa Ser Xaa Arg Leu Arg Leu Xaa Xaa Thr Tyr Xaa Tyr as shownin SEQ ID NO: 1, wherein Xaa at position 5 is Gly or Ala; Xaa atposition 7 is Ser or Gly; Xaa at position 12 is Gly, Ala or Pro; Xaa atposition 13 is Asp or Gly; Xaa at position 16 is Asp or Glu; and whereinsaid VEGF-binding component is capable of blocking the interaction ofhuman recombinant VEGF165 with the human recombinant VEGFR-2 with aninhibition rate of ≧60%.
 3. A bispecific binding molecule of claim 2,wherein said CDR3 has a sequence selected from SEQ ID NO: 2SRAYGSSRLRLGDTYDY, SEQ ID NO: 3 SRAYGSSRLRLADTYDY; SEQ ID NO: 4SRAYGSSRLRLADTYEY; SEQ ID NO: 5 SRAYGSGRLRLADTYDY; SEQ ID NO: 6SRAYASSRLRLADTYDY; SEQ ID NO: 7 SRAYGSSRLRLPDTYDY; SEQ ID NO: 8SRAYGSSRLRLPGTYDY.


4. A bispecific binding molecule of claim 3, wherein said VEGF-bindingcomponent comprises one or more immunoglobulin single variable domainseach containing a. a CDR3 with an amino acid sequence selected from afirst group of sequences shown in SEQ ID NO: 2 to 8; b. a CDR1 and aCDR2 with an amino acid sequences that is contained, as indicated inTable 3, in a sequence selected from a second group of sequences shownin SEQ ID NOs: 9 to 46, wherein said second sequence contains therespective CDR3 in said selected sequence according to a).
 5. Abispecific binding molecule of claim 4, wherein said one or moreimmunoglobulin single variable domains are VHHs.
 6. A bispecific bindingmolecule of claim 5, wherein said one or more VHHs have amino acidsequences selected from the amino acid sequences shown in SEQ ID NOs:9-46.
 7. A bispecific binding molecule of claim 6, which comprises oneor more VHHs having amino acid sequences selected from SEQ ID NO: 15,SEQ ID NO: 18 and SEQ ID NO:
 25. 8. A bispecific binding molecule, theVEGF-binding component of which has been obtained by affinity maturationand/or sequence optimization of a VHH defined in claim
 7. 9. Abispecific binding molecule according to claim 8, the VEGF-bindingcomponent of which has been obtained by sequence optimization of a VHHhaving an amino acid sequence shown in SEQ ID NO:
 18. 10. A bispecificbinding molecule according to claim 9, the VEGF-binding component ofwhich having an amino acid sequence selected from sequences shown in SEQID NOs: 47-57.
 11. A bispecific binding molecule according to claim 5,the VEGF-binding component of which comprising two or more VHHs, whichare a. identical VHHs that are capable of blocking the interactionbetween recombinant human VEGF and the recombinant human VEGFR-2 with aninhibition rate of 60% or b. different VHHs that bind to non-overlappingepitopes of VEGF, wherein at least one VHH is capable of blocking theinteraction between recombinant human VEGF and the recombinant humanVEGFR-2 with an inhibition rate of z 60% and wherein at least one VHH iscapable of blocking said interaction with an inhibition rate of ≧60%.12. A bispecific binding molecule according to claim 11, wherein saididentical VHHs a) are selected from VHHs having amino acid sequencesshown in SEQ ID NOs: 9-46 or VHHs that have been obtained by affinitymaturation and/or sequence optimization of such VHH.
 13. A bispecificbinding molecule according to claim 12, wherein said VHH is selectedfrom VHHs having the amino acid shown in SEQ ID NO: 18 or SEQ ID NO:47-57.
 14. A bispecific binding molecule according to claim 13comprising two VHHs each having the amino acid sequence shown in SEQ IDNO:
 57. 15. A bispecific binding molecule according to claim 14, whereina. said one or more VHHs with an inhibition rate of z 60% are selectedfrom i. VHHs having an amino acid sequence selected from amino acidsequences shown in SEQ ID NOs: 9-46 or ii. VHHs that have been obtainedby affinity maturation and/or sequence optimization of such VHHs, andwherein b. said one or more VHHs with an inhibition rate of 60% areselected from i. SEQ ID NOs: 58-124 or ii. VHHs that have been obtainedby affinity maturation and/or sequence optimization of such VHH.
 16. Abispecific binding molecule according to claim 15, wherein two VHHs arecontained in polypeptides with amino acid sequences shown in SEQ ID NOs:128-168, separated by linker sequences as indicated in Table
 13. 17. Abispecific binding molecule according to claim 16, wherein said VHH a)i. has an amino acid sequence shown in SEQ ID NO: 18 and said VHH b) i.has an amino acid sequence shown in SEQ ID NO:
 64. 18. A bispecificbinding molecule according to claim 17, wherein said VHHs according toa) ii) are selected from VHHs having an amino acid sequence shown in SEQID NOs: 47-57 and wherein said VHHs according to b) ii) are selectedfrom VHHs having an amino acid sequence shown in SEQ ID NOs: 125-127.19. A bispecific binding molecule according to claim 18, comprising twoVHHs, one of them having the amino acid shown in SEQ ID NO: 57 and oneof them having the amino acid shown in SEQ ID NO:
 127. 20. Thebispecific binding molecule of claim 1, comprising an Ang2-bindingcomponent comprising at least a variable domain with four frameworkregions and three complementarity determining regions CDR1, CDR2 andCDR3, respectively, wherein said CDR3 has an amino acid sequenceselected from amino acid sequences shown in SEQ IDs NOs: 226, 229, 232,235, 238, 241, 244, 247, 250, or
 253. 21. The bispecific bindingmolecule of claim 20, the Ang2-binding component of which is an isolatedimmunoglobulin single variable domain or a polypeptide containing one ormore of said immunoglobulin single variable domains, wherein saidimmunoglobulin single variable domain consists of four framework regionsand three complementarity determining regions CDR1, CDR2 and CDR3,respectively, and wherein said CDR3 has an amino acid sequence selectedfrom amino acid sequences shown in SEQ IDs NOs: 226, 229, 232, 235, 238,241, 244, 247, 250, or
 253. 22. The bispecific binding molecule of claim21, wherein said one or more immunoglobulin single variable domaincontain a. a CDR3 with an amino acid sequence selected from a firstgroup of amino acid sequences shown in SEQ ID NOs: SEQ IDs NOs: 226,229, 232, 235, 238, 241, 244, 247, 250, or 253 (Table 49); b. a CDR1with an amino acid sequences that is contained, as indicated in Table36-A, 38-A, 41-A, or 45-A, as partial sequence in a sequence selectedfrom a second group of amino acid sequences shown SEQ ID NOs: 224, 227,230, 233, 236, 239, 242, 245, 248, or 251 (Table 49); c. a CDR2 with anamino acid sequences that is contained, as indicated in Table 36-A,38-A, 41-A, or 45-A, as partial sequence in a sequence selected from asecond group of amino acid sequences shown SEQ ID NOs:225, 228, 231,234, 237, 240, 243, 246, 249, or 252 (Table 49).
 23. The bispecificbinding molecule of claim 20, wherein said one or more immunoglobulinsingle variable domains are VHHs.
 24. The bispecific binding molecule ofclaim 23, wherein said one or more VHHs have an amino acid sequenceselected from amino acid sequences shown in SEQ ID NOs: 214, 215, 216,217, 218, 219, 220, 221, 222, or
 223. 25. An immunoglobulin singlevariable domain which has been obtained by affinity maturation of animmunoglobulin single variable domain as defined in claim
 22. 26. A VHHwhich has been obtained by affinity maturation of a VHH as defined inclaim
 24. 27. An Ang2-binding VHH with an amino acid sequence selectedfrom acid sequences shown in SEQ ID NOs: 214, 215, 216, 217, 218, 219,220, 221, 222, or
 223. 28. An immunoglobulin single variable domainwhich has been obtained by humanization of a VHH defined in claim 27.29. An immunoglobulin single variable domain which has been obtained byhumanization of an immunoglobulin single variable domain as defined inclaim
 22. 30. The binding molecule of claim 1, the serum albumin bindingcomponent of which is an isolated immunoglobulin single variable domainor a polypeptide containing one or more of said immunoglobulin singlevariable domains, wherein said immunoglobulin single variable domainconsists of four framework regions and three complementarity determiningregions CDR1, CDR2 and CDR3, respectively, and wherein said CDR3 has anamino acid sequence selected from amino acid sequences shown in SEQ IDNOs: 257, 260, 263, 266, 269, 272, or
 275. 31. The binding molecule ofclaim 30, wherein said one or more immunoglobulin single variable domaincontain a. a CDR3 with an amino acid sequence selected from a firstgroup of amino acid sequences shown in SEQ ID NOs: SEQ IDs NOs: 257,260, 263, 266, 269, 272, or 275; b. a CDR1 with an amino acid sequencesselected from a second group of amino acid sequences shown SEQ ID NOs:255, 258, 261, 264, 267, 270, or 273; c. a CDR2 with an amino acidsequences selected from a second group of amino acid sequences shown SEQID NOs: 256, 259, 262, 265, 268, 271, or
 274. 32. The bispecific bindingmolecule of claim 30, wherein said one or more immunoglobulin singlevariable domains are VHHs.
 33. The bispecific binding molecule of claim32, wherein said one or more VHHs have an amino acid sequence shown inSEQ ID NOs: 98 or
 254. 34. The bispecific binding molecule of claim 1having the amino acid sequence selected from amino acid sequences shownin SEQ ID NOs: 180-213.
 35. A nucleic acid molecule encoding abispecific binding molecule of claim 1 or a vector containing same. 36.A host cell comprising a nucleic acid molecule of claim
 35. 37. Apharmaceutical composition comprising at least one bispecific bindingmolecule of claim 1 as the active ingredient.
 38. A method of treating adisease that is associated with VEGF- and/or Ang2-mediated effects onangiogenesis comprising administering to a patient an effective amountof a pharmaceutical composition according to claim
 37. 39. The method ofclaim 38 wherein the disease is selected from cancer and cancerousdiseases.
 40. The method of claim 38 wherein the disease is eyediseases.